Compositions, methods and uses for dengue virus serotype-4 constructs

ABSTRACT

Embodiments herein report compositions, methods and uses for dengue-4 (DENV-4) virus constructs. Some embodiments concern a composition that includes, but is not limited to, DENV-4 virus constructs alone or in combination with other constructs, can be used in a vaccine composition to induce an immune response in a subject. In certain embodiments, compositions can include constructs of more than one serotypes of dengue virus, such as dengue-1 virus, dengue-2 virus, or dengue-3 virus in combination with DENV-4 virus constructs disclosed herein. In other embodiments, DENV-4 constructs disclosed herein can be combined in a composition with other flavivirus constructs to generate a vaccine against more than one flavivirus. Other embodiments provide methods and uses for DENV-4 virus constructs in vaccine compositions that when administered to a subject induce an immune response in the subject against DENV-4 that is improved by modified constructs compared to other vaccine compositions.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application No.61/724,190 filed Nov. 8, 2012 and U.S. Provisional Application No.61/788,536 filed Mar. 15, 2013. All prior applications are incorporatedherein in their entirety by reference for all purposes.

FEDERALLY FUNDED RESEARCH

Some embodiments disclosed herein were supported in part by grant numberR43 AI084291-01 from the National Institutes of Health. The U.S.Government may have certain rights to practice the subject invention.

FIELD

Embodiments herein report compositions, methods and uses fordengue-virus 4 (DENV-4) constructs. Some embodiments concern acomposition that includes, but is not limited to, DENV-4 virusconstructs that alone or in combination with other agents can be used ina vaccine composition. In certain embodiments, compositions can includechimera constructs of more than one serotypes of dengue virus, such asdengue-1 (DEN-1) virus, dengue-2 (DEN-2) virus, or dengue-3 (DEN-3)virus in combination with DENV-4 virus chimera constructs in di-, tri ortetravalent formulations. In other embodiments, DENV-4 chimeraconstructs (dengue-dengue chimeras) disclosed herein can be combinedwith other flavivirus constructs. Certain embodiments include DENV-4chimeric constructs having components of other dengue serotypes, such asstructural elements. Other embodiments provide methods and uses forDENV-4 virus chimera constructs in vaccine compositions that whenadministered to a subject induces an immune response in the subjectagainst DENV-4 that is improved compared to other constructs.

BACKGROUND

Infection with dengue virus can lead to a painful fever of varyingseverity. To date, five serotypes of dengue virus have been identified:dengue-1 (DEN-1), dengue-2 (DEN-2), or dengue-3 (DEN-3), dengue-4(DENV-4) and dengue-5 (DEN-5). Dengue fever is caused by infection of adengue virus. Dengue virus serotypes 1-4 can also cause denguehemorrhagic fever (DHF), and dengue shock syndrome (DSS). The mostsevere consequences of infection, DHF and DSS, can be life threatening.Dengue viruses cause 50-100 million cases of debilitating dengue fever,500,000 cases of DHF/DSS, and more than 20,000 deaths each year. Todate, there is no effective vaccine to protect against dengue fever andno drug treatment for the disease. Mosquito control efforts have beenineffective in preventing dengue outbreaks in endemic areas or inpreventing further geographic spread of the disease. It is estimatedthat 3.5 billion people are threatened by infection with dengue virus.In addition, dengue virus is a leading cause of fever in travelers toendemic areas, such as Asia, Central and South America, and theCaribbean.

All four dengue virus serotypes are endemic throughout the tropical andsubtropical regions of the world and constitute the most significantmosquito-borne viral threat to humans worldwide. Dengue viruses aretransmitted to humans primarily by Aedes aegypti mosquitoes. Infectionwith one dengue virus serotype results in life-long protection fromre-infection by that serotype, but does not prevent secondary infectionby one of the other three dengue virus serotypes. In fact, previousinfection with one dengue virus serotype can lead to an increased riskof severe disease (DHF/DSS) upon secondary infection with a differentserotype. The development of an effective vaccine represents animportant approach to the prevention and control of this global emergingdisease.

SUMMARY

Embodiments herein concern compositions, methods and uses of DENVchimera constructs, for example DENV-4. In some embodiments, acomposition can include DENV-4 virus chimera constructs alone or incombination with other dengue virus serotype constructs or live,attenuated dengue viruses of the same or other serotypes or otherflavivirus constructs capable of inducing an immune response to a targetvirus (e.g. dengue virus). Other embodiments can include a compositionof a live, attenuated virus construct against DENV-4 and optionally, oneor more live, attenuated viral constructs against DEN-1, DEN-2 andDEN-3. In other embodiments, an immunogenic composition is provided thatincludes a DENV-4 live, attenuated chimeric virus construct with strongimmunogenicity when introduced to a subject. In accordance with theseembodiments, these live, attenuated viral constructs can be used aloneor in combination with one or more other DEN-1, DEN-2 and DEN-3constructs, and a pharmaceutically acceptable excipient to generate avaccine formulation against dengue virus serotypes. In certainembodiments, monovalent, bivalent, trivalent or tetravalentpharmaceutically effective formulations against one or more dengueviruses are generated. In certain embodiments, an immunogeniccomposition can include one or more of DEN-1, DEN-2, DEN-3 dengue-denguechimeric constructs in combination with a chimeric DENV-4 constructdisclosed herein.

In certain embodiments, an immunogenic composition including a DENV-4construct of the present invention in combination with one or more ofDEN-1, DEN-2 and DEN-3 can be used to confer simultaneous protectionagainst two or more dengue virus serotypes in a single vaccineadministration. In other embodiments, an immunogenic compositionincluding DEN-1, DEN-2, DEN-3 and modified or mutated DENV-4 constructsof embodiments disclosed herein can be administered to a subject toinduce improved immunogenic responses against each dengue virus serotypeand where immune response interference to DENV-4 is reduced.

In certain embodiments, DENV-4 constructs can include a dengue-denguechimeric construct having adaptive mutations in the structural ornon-structural regions of DENV-4. In other embodiments, DENV-4constructs can include a backbone of another dengue virus serotype,DEN-1, DEN-2 or DEN-3. In yet other embodiments, a chimeric constructcan include a DEN-2 backbone where DENV-4 structural or non-structuralregions of DENV-4 are substituted for DEN-2 structural or non-structuralregions. In accordance with these embodiments, a DEN-2 backbone caninclude any live attenuated DEN-2 virus. In other embodiments, a DEN-2backbone can include live attenuated DEN-2 PDK-53 virus as a backbonewhere the live attenuated DEN-2 PDK virus further includes structuralproteins of one or more of prM (premembrane) and E (envelope) structuralproteins of DENV-4. In addition, a DEN-2 PDK-53 backbone can includeadditional mutations or reversions of mutations of DEN-2 PDK-53generating a novel construct in order to enhance in vitro growth, or invivo the immune response to DENV-4 in a subject upon administration.

In some embodiments, a current dengue chimeric construct denoted asDENVax-4 strain (SEQ ID NO:21) was modified to contain a capsid/PrMjunction of the DEN-2 backbone to be more genetically similar to that ofDENV-4 instead of DEN-2 in order to improve replication efficiency ofthe virus both in vitro for production and in vivo as a construct of usefor inducing an immune response to DENV-4. The current strain of DEN-4,DENVax-4, has a capsid/PrM sequence that is identical to DEN-2 insteadof DEN-4, possibly creating an inefficient transcription and translationfrom the genomic RNA, which is different than that of wild type DENV-4.

In some embodiments, structural protein genes can include prM and Egenes of DENV-4 on another dengue virus backbone (e.g. dengue-2, DEN-2PDK-53), making a dengue-dengue chimera. For example, a DEN-4 construct,in certain embodiments can include those construct termed DENVax-4e(Capsid 107 Cysteine to Tyrosine; DenVax-4b backbone, modifications atCapsid/prM junction), DENVax-4f (where the PDK-53 backbone NS2A and NS4Amutations are reverted to that of 16681) or DENVax-4h (Envelope 417 Gluto Lys) (see for example FIG. 4) where for certain constructs the DEN-2PDK-53 backbone has one or more reversions to wild-type DEN-2 (e.g. inthe non-coding region (NCR) or a non-structural region (NS2 etc.)) andone or more mutations in the DENV-4 structural region (e.g. prM or E),while encoding one or more structural proteins of DENV-4 (e.g. strain1036). A modified DENV-4 construct disclosed herein can include amodified attenuated DEN-2 PDK-53 backbone, having one or more modifiedstructural proteins of DENV-4 strain 1036. In some embodiments, one ormore mutations present in live, attenuated DEN-2 PDK-53 virus can bereverted back to a wildtype nucleic acid (which may be a silentmutation) or another nucleic acid to produce constructs herein thatgenerate a modified DEN-2/DENV-4 construct having increased replicationability and immunogenicity without affecting its attenuation or safetybut may affect growth and/or replication of the DEN-4 virus. In certainembodiment, the reversions may lead to increased growth and/orreplication.

In other embodiments, a modified DENV-4 construct can incorporatemutations introduced to one or more structural regions and/ornon-structural regions of DENV-4 in order to generate constructsinducing an improved immunological response while maintaining safety andviral attenuation. For example, a modified or mutated dengue-denguechimera of DEN-2/DENV-4 may contain mutations at one or morenon-structural regions of a DEN-2 PDK-53 backbone, such as NS2A, andNS4A, and/or mutations at 5′ non-coding region (5′NCR). In anotherembodiment, a modified DENV-4 chimera construct can include NS2A andNS4A of DEN-2 16681 by reverting mutations at NS2A and NS4A of PDK-53(e.g. an M-L substitution at NS4A). Some embodiments include a modifiedDENV-4 chimera construct having 5′NCR, NS2A and NS4A of DEN-2 16681 byreverting corresponding mutations in the DEN-2 PDK-53 backbone of atarget construct. Other embodiments can include a modified DENV-4chimera construct having 5′NCR of DEN-2 16681 by reverting correspondingmutations in the DEN-2 PDK-53 backbone. A modified DEN-4 chimeraconstruct can also include DEN-2 PDK-53 backbone, and encode one or morestructural proteins of DEN-4 strain H241. It is contemplated that, toinduce an immune response, any DEN-4 structural protein can besubstituted for structural regions of a chimeric virus containing adengue-2 serotype backbone (e.g. PDK-53 or modified PDK-53). In someembodiments, a modified DEN-4 construct contains live attenuated DEN-2PDK-53 as a backbone, and DEN-4 structural proteins where mutations canbe introduced to modify structural regions of a DEN-4 (e.g. strain1036).

In other embodiments, mutations can be introduced to capsid/prM junctionamino acid sequences of a DENV virus in order to increase immunogenicityof a construct containing such a mutation. For example, a mutation inDEN-4 can be a Cys-Tyr mutation at capsid position 107 of the DEN-4. Inother embodiments, it is contemplated that the cysteine in position 107can be mutated to any other aromatic amino acid with a hydrophobic sidechain (see for example DEN-4e). Other DEN-2 PDK-53 reversion of achimeric construct can be found in NS2A or NS4A. Yet other embodimentsinclude a DEN-4 construct where a DEN-2 backbone comprises PDK-53 (MVS,SEQ ID NO:21) where amino acid positions 102-107 of the capsid region ofPDK-53 are converted to a homologous DEN-4 counterpart amino acid togenerate DENV-4b (see for example, FIG. 4). These backbone constructscan then further comprise a cysteine in the capsid region to aromaticamino acid in position (e.g. tyrosine, tryptophan etc). In certainembodiments, this construct is represented by SEQ ID NO:22 or SEQ IDNO:23.

Other DENV-4 constructs disclosed herein can include an amino acidsubstitution at Envelope position 417. For example, DEN-4 strain 1036strain sequence or equivalent strain position thereof where a PDK-53(MVS DEN2/4, SEQ ID NO:21) backbone of Dengue-2 with DEN-4 structuralproteins is provided. Embodiments include further mutating Envelopeposition 417 from a negative to a positively charged side-chain aminoacid (e.g. lysine). It is contemplated that any charged side chain willprovide increased immunogenicity of the DEN-4 construct withoutaffecting its safety or attenuation. In certain embodiments, thisconstruct is represented by SEQ ID NO:24 or SEQ ID NO:25.

In certain embodiments, DEN-2 PDK-53 reversions of a chimeric DENVconstruct have the 5′ NC, NS1 and NS3 mutations found in DEN-2 PDK-53MVS while having other reversions or mutations. It has been demonstratedthat these three mutations can be important for attenuation (e.g. smallplaque size, reduced growth rate, lower titer, increased temperaturesensitivity and decreased neurovirulence compared to a control).

In other embodiments, DEN-2 PDK-53 genome backbones can be used togenerate chimeric constructs of DEN-1 and DEN-3, where one or morestructural protein genes of DEN-2 PDK-53 genome can be replaced by oneor more structural protein genes of DEN-1 and DEN-3. These constructscan include a combination of both DEN-1 and DEN-3 in a single chimerahaving a DEN-2 PDK-53 backbone. In some embodiments, a structuralprotein can be the C, prM or E protein of DEN-1 and/or DEN-3. In certainembodiments, structural protein genes include the prM and E genes ofDEN-1 or DEN-3. These hybrid/chimeric viruses express the surfaceantigens of DEN-1, DEN-3 or DENV-4 while retaining the attenuationphenotypes of the parent DEN-2. In certain embodiments, these constructscan be represented by SEQ ID NO:15, DEN-2/DEN-1 and SEQ ID NO: 19,DEN-2/DEN-3 where these constructs can be used in di-, tri ortetravalent compositions disclosed herein.

In some embodiment, constructs disclosed herein can include chimericconstructs of DENV-4, DEN-2, DEN-1, and DEN-3 expressing surfaceantigens of DEN-1, DEN-3 and DENV-4 using attenuated DEN-2 PDK-53 virusas a backbone.

Some embodiments disclose methods for making modified or mutated DENV-4constructs of use in any vaccine composition including, but not limitedto, a single vaccine composition having only DENV-4 constructs, amixture single vaccine composition capable of inducing an immuneresponse against two or more dengue virus serotypes, a mixture singlevaccine composition having chimeric (and non-chimeric) constructsdisclosed herein in combination with other flavivirus constructs capablein inducing an immune response to a flavivirus as well as one or moredengue virus serotypes that include DENV-4.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments. Some embodimentsmay be better understood by reference to one or more of these drawingsalone or in combination with the detailed description of specificembodiments presented.

FIGS. 1A-1B illustrates (A) a representative schematic diagram ofstructural and non-structural genes of DEN-2 PDK-53, and (B) exemplaryresults of certain chimeric dengue virus constructs of some embodimentsdisclosed herein expressing structural proteins of different denguevirus serotypes respectively in the DEN-2 PDK-53 backbone.

FIG. 2 is a table that illustrates an exemplary experimental design foranalyzing certain vaccines including DENV-4 chimeric constructs of someembodiments disclosed herein in non-human primates.

FIG. 3 represents an exemplary graph of an experiment related toproduction of DENV-4 neutralizing antibody titers produced by an animalmodel immunized with a vaccine composition having DENV-4 chimeric viralconstructs of certain embodiments disclosed herein.

FIG. 4 represents a schematic of certain DENV-4 chimera constructsdisclosed in some embodiments herein and used in vaccine formulationsalone or in combination with other live, attenuated virus constructs.

FIG. 5 is a table illustrating some exemplary data of analysis of plaquephenotypes, Vero cell titers and mosquito growth of some DENV-4 virusconstructs of certain embodiments disclosed herein.

FIGS. 6A-6F are photographs providing exemplary phenotypes of ImmunoFoci(plaques) results of an exemplary experiment using certain DENV-4chimera constructs including DENV-4 wild type (A), DENVax-4 (B),DENVax-4e (C), DENVax-4h (D), DENVax-4i (E), and DENVax-4j (F).

FIG. 7 is a graphic representation of viral titers over time of variousDENV-4 chimeric constructs after growth in mammalian cells, illustratedas “Days Post Infection.”

FIG. 8 is a graphic representation of viral titers over time of variousDENV-4 chimeric constructs after growth in mosquito cells illustrated as“Days Post Infection.”

FIG. 9 is a flow chart representing an acceptable animal model used totest various DENV-4 chimeric constructs to induce an immune response toDENV-4 in the animal model.

FIG. 10 illustrates an alignment of various DENV-4 chimeric constructs,illustrating common sequences and exemplary mutations and/or reversions.

FIG. 11 illustrates an alignment of various DENV-4 chimeric constructs,illustrating common sequences and exemplary mutations and/or reversions.

FIG. 12 illustrates a schematic representation of a live, attenuatedvirus construct of DEN-2 virus.

FIG. 13 illustrates an exemplary schematic of cloning of various DENV-4constructs disclosed herein.

FIG. 14 represents a graph comparing various DENV-4 constructs growth inmosquito cells.

FIG. 15 represents an exemplary histogram plot comparing immunogenicityin mice of various DENV-4 constructs disclosed herein.

FIG. 16 represents an exemplary graph illustrating efficacy usingvarious DEN-4 constructs in mice to protect immunized mice againstchallenge by wt-DENV, represented as survival.

FIG. 17 represents an alignment of the flavivirus envelope protein.

FIG. 18 represents a schematic of a DENV virus including open readingframes for proteins of DENV.

FIG. 19 represents a graphic illustration of titer achieved after growthof various DEN-4 constructs in a mammalian cell line contemplated of useherein.

FIG. 20 represents various levels of neutralizing antibodies aftervaccination with various dengue constructs in non-human primates postvaccination, represented as days post vaccination.

FIG. 21 represents various levels of neutralizing antibodies aftervaccination with various dengue constructs in non-human primates postvaccination, represented as days post vaccination.

FIG. 22 represents various levels of dengue constructs in a mouse postvaccination, represented as days post vaccination.

FIG. 23 represents various levels of dengue constructs in a mouse postvaccination, represented as days post vaccination.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein the specification, “subject” or “subjects” may include,but are not limited to, mammals such as humans (e.g. adults andjuveniles) or mammals, domesticated or wild, for example dogs, cats,other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets,rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zooanimals.

As used herein, the terms “chimeric construct,” “virus chimera,”“chimeric virus,” “flavivirus chimera” and “chimeric flavivirus” canmean a construct comprising a portion of the nucleotide sequence of adengue-2 virus and further nucleotide sequence that is not from dengue-2virus or is from a different dengue virus serotype or a differentflavivirus. A “dengue chimera” comprises at least two different denguevirus serotypes. Examples of other dengue viruses or flavivirusesinclude, but are not limited to, sequences from dengue-1 virus, dengue-3virus, dengue-4 virus, West Nile virus, Japanese encephalitis virus, St.Louis encephalitis virus, tick-borne encephalitis virus, yellow fevervirus and any combination thereof.

As used herein, “nucleic acid chimera” can mean a construct disclosedherein including nucleic acid sequences comprising a portion of thenucleotide sequence of a dengue-2 virus and further one or morenucleotide sequences are not of the same origin as the nucleotidesequence of the dengue-2 virus. Correspondingly, any chimericflavivirus, any dengue chimera or flavivirus chimera disclosed hereincan be recognized as an example of a nucleic acid chimera.

DESCRIPTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments. It will be obviousto one skilled in the art that practicing the various embodiments doesnot require the employment of all or even some of the specific detailsoutlined herein, but rather that concentrations, times and otherspecific details may be modified through routine experimentation. Insome cases, well-known methods or components have not been included inthe description.

In accordance with embodiments of the present invention, there may beemployed conventional molecular biology, protein chemistry,microbiology, and recombinant DNA techniques within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,Second Edition 1989, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed., 1986).

Embodiments herein concern compositions, methods and uses for inducingimproved immune responses against DEN-4 alone or in combination with oneor more agents for inducing immune responses against other dengue virusserotypes or flaviviruses in a subject. In accordance with theseembodiments, live, attenuated dengue viruses and nucleic acid chimerasof DEN-4 are generated and used in immunogenic compositions disclosedherein. Some embodiments concern modified or mutated DEN-4 constructs.Some embodiments concern introducing mutations and/or reversions intoDEN-4 chimeric constructs to modify the amino acid sequence of thechimeric construct. Some embodiments concern introducing mutationsand/or reversions into the DEN-2 PDK-53 backbone constructs to modifythe amino acid sequence or RNA sequence of the chimeric construct. Incertain embodiments, mutations and/or reversions into DEN-4 chimericconstructs to modify the amino acid sequence of the chimeric constructcan include mutations upstream of the C/prM cleavage site of a knownchimeric construct referred to as DENVax-4 (SEQ ID NO: 21) by techniquesincluding, but not limited to mutagenesis.

Embodiments herein concern compositions, methods and uses of DENV-4virus chimera constructs. In some embodiments, a composition can includeDENV-4 virus chimera constructs alone or in combination with otherdengue virus serotype constructs or live, attenuated dengue viruses ofthe same or other serotypes or other flavivirus constructs capable ofinducing an immune response to a target virus (e.g. dengue virus orother flavivirus). Other embodiments can include a composition of alive, attenuated virus construct against DENV-4 and optionally, one ormore live, attenuated viral constructs against DEN-1, DEN-2 and DEN-3.In yet other embodiments, an immunogenic composition is provided thatincludes a DENV-4 live, attenuated chimeric virus constructs withincreased immunogenicity compared to other known constructs whenintroduced to a subject. In accordance with these embodiments, theselive, attenuated viral chimera constructs can be used alone or incombination with one or more other DEN-1, DEN-2 and DEN-3 constructs(e.g. live, attenuated viruses or chimeras), and a pharmaceuticallyacceptable excipient to generate a vaccine formulation against one ormore dengue virus serotypes. In certain embodiments, monovalent,bivalent, trivalent or tetravalent pharmaceutically effectiveformulations against one or more dengue viruses are generated. Incertain embodiments, an immunogenic composition can include one or moreof DEN-1, DEN-2, DEN-3 dengue-dengue chimeric constructs or live,attenuated dengue virus in combination with a chimeric DENV-4 constructdisclosed herein.

In certain embodiments, an immunogenic composition including a DENV-4construct of the present invention in combination with one or more ofDEN-1, DEN-2 and DEN-3 can be used to confer simultaneous protectionagainst two or more dengue virus serotypes in a single vaccineadministration. In other embodiments, an immunogenic compositionincluding DEN-1, DEN-2, DEN-3 and modified or mutated DENV-4 constructsof embodiments disclosed herein can be administered to a subject toinduce improved immunogenic responses against each dengue virus serotypeand where immune response interference to DENV-4 is reduced.

In certain embodiments, DENV constructs can include a dengue-denguechimeric construct having adaptive mutations in the structural ornon-structural regions of the backbone (PDK-53) or structural regions ofDEN-4. In other embodiments, DENV-4 constructs can include a backbone ofanother dengue virus serotype, DEN-1, DEN-2 or DEN-3. In yet otherembodiments, a chimeric construct can include a DEN-2 backbone whereDENV-4 structural or non-structural regions of DENV-4 are substitutedfor DEN-2 structural and/or non-structural regions. In accordance withthese embodiments, a DEN-2 backbone can include any live, attenuatedDEN-2 virus having safety and efficacy while inducing an immune responseto DEN-2. In other embodiments, a DEN-2 backbone can include live,attenuated DEN-2 PDK-53 (53 passages in primary dog kidney cells (PDK))or derived from DEN-16681 strain virus as a backbone where the live,attenuated DEN-2 PDK-53 virus further includes structural proteins ofone or more of prM (premembrane) and E (envelope) structural proteins ofDENV-4. In addition, a DEN-2 PDK-53 backbone can include additionalmutations or reversions of mutations of DEN-2 PDK-53 in order to enhancethe immune response to DENV-4 in a subject upon administration (see forexample FIG. 4).

In some embodiments, structural protein genes can include prM and Egenes of DENV-4 on another dengue virus backbone, making a dengue-denguechimera. For example, a DENV-4 construct, in certain embodiments caninclude those construct termed DENVax-4e, DENVax-4f, or DENVax-4h (seefor example FIG. 4) where the DEN-2 PDK-53 backbone has one or morereversions to wild-type DEN-2 amino acids (e.g. in the non-coding region(NCR) or a non-structural region (NS1 etc.)) and one or more mutationsin the DENV-4 structural region (e.g. prM or E), while encoding one ormore structural proteins of DENV-4 (e.g. strain 1036). A modified DENV-4construct disclosed herein can include a modified attenuated DEN-2PDK-53 backbone, having one or more modified structural regions ofDENV-4 strain 1036. In some embodiments, one or more mutations presentin live, attenuated DEN-2 PDK-53 virus can be reverted back to a controlamino acid or another amino acid to produce constructs herein thatgenerate a modified DEN-2/DENV-4 chimeric construct (when compared toMVS sequence SEQ ID. NO: 21) having increased immunogenicity withoutaffecting its safety or attenuation but may affect in vitro growthand/or replication of the DEN-4 virus. In certain embodiment, thereversions may lead to increased growth and/or replication.

In other embodiments, a modified DENV-4 construct can incorporatemutations introduced to one or more structural regions and/ornon-structural regions of DENV-4 in order to generate constructsinducing an improved immunological response while maintaining safety andviral attenuation. For example, a modified or mutated dengue-denguechimera of DEN-2/DENV-4 may contain mutations at one or morenon-structural regions of a DEN-2 PDK-53 backbone, such as NS2A, andNS4A, and/or mutations at 5′ non-coding region (5′NCR). In anotherembodiment, a modified DENV-4 chimera construct can include NS2A andNS4A of DEN-2 16681 by reverting mutations at NS2A and NS4A of PDK-53(e.g. an M-L substitution at NS4A). Some embodiments include a modifiedDENV-4 chimera construct having 5′NCR, NS2A and NS4A of DEN-2 16681 byreverting corresponding mutations in the DEN-2 PDK-53 backbone of atarget construct. Other embodiments can include a modified DENV-4chimera construct having 5′NCR of DEN-2 16681 by reverting correspondingmutations in the DEN-2 PDK-53 backbone. A modified DENV-4 chimeraconstruct can also include DEN-2 PDK-53 backbone, and encode one or morestructural proteins of DENV-4 strain H241.

It is contemplated that DENV-4 structural proteins can substitute forstructural or non-structural regions of a dengue-2 serotype backbone(e.g. PDK-53 or modified PDK-53 identified herein) In some embodiments,a modified DENV-4 construct contains live attenuated modified DEN-2PDK-53 as a backbone, and DENV-4 structural proteins where mutations canbe introduced to modify structural regions of a DENV-4 (e.g. strain1036). In some embodiments, mutations can be introduced to capsid/prMjunction amino acid sequences of DENV-4 in order to increase replicationand/or immunogenicity of a construct containing such a mutation. Forexample, a mutation in DENV-4 can be a C-Y mutation at capsid position107 of the DENV-4 (see for example, DENVax-4e). In accordance with theseembodiments, a cysteine can be mutated to an aromatic amino acid (e.g.tyrosine or other) on a modified PDK-53 backbone (DENV-4b). Othermutations can include an amino acid substitution at Envelope position417 (glutamic acid, E) in a DENV-4 1036 strain sequence (see forexample, DENVax-4h) or equivalent position thereof in another DENV-4,where a negative amino acid is replaced by a positive amino acid with acharged side chain (e.g. lysine, arginine, histidine etc.). Other DEN-2PDK-53 reversion of a chimeric construct can be found in the NS2A orNS4A regions.

In other embodiments, DEN-2 PDK-53 genome backbones can be used togenerate chimeric dengue virus constructs of DEN-1 and DEN-3, where oneor more structural or non-structural protein genes of DEN-2 PDK-53genome can be replaced by one or more structural protein ornon-structural genes of DEN-1 and DEN-3. These constructs can include acombination of both DEN-1 and DEN-3 structural or non-structural genesin a single chimera having a DEN-2 PDK-53 backbone. In some embodiments,a structural protein can be the C, prM or E protein of DEN-1 and/orDEN-3. In certain embodiments, structural protein genes include the prMand E genes of DEN-1 or DEN-3 or a combination thereof. Thesehybrid/chimeric viruses can express surface antigens of DEN-1, DEN-3 orDENV-4 while retaining the attenuation phenotypes of the parent DEN-2.

In some embodiment, constructs disclosed herein can include chimericconstructs of DENV-4, DEN-2, DEN-1, and DEN-3 expressing surfaceantigens of DEN-1, DEN-3 and DENV-4 using attenuated DEN-2 PDK-53 orlive, attenuated DEN-2 16681 virus (or a dengue-2 virus with one or morereversion of any of the mutations found in dengue-2 serotype PDK-53 backto its wildtype 16681 virus) as a backbone. In addition, constructs thatare part of a pharmaceutical composition can include other agents suchas other live, attenuated viruses (e.g. DEN-2, other flaviviruses).Further, other agents of use in these compositions can include otherpharmaceutically acceptable anti-viral agents, adjuvants or stabilizingagents to reduce degradation of the live, attenuated viruses.

Some embodiments herein disclose methods for making modified or mutatedDENV-4 constructs of use in any vaccine composition against DENV-4including, but not limited to, a single vaccine composition having onlyDENV-4 constructs, a mixture of dengue virus constructs of a singlevaccine composition capable of inducing an immune response against twoor more dengue virus serotypes, a mixture in a single vaccinecomposition having chimeric (and non-chimeric) constructs disclosedherein in combination with other flavivirus constructs capable ininducing an immune response to a different flavivirus (e.g. yellowfever, West Nile, Japanese encephalitis etc.) as well as one or moredengue virus serotypes that include DENV-4.

In other embodiments, other combinations are contemplated of use withDENV-4 constructs disclosed herein. For example, a dengue virus serotype1 wild-type virus passaged in PDK cells 13 times is designated as DEN-1PDK-13 virus. Other vaccine candidates are DEN-2 PDK-53, DEN-3PGMK-30/FRhL-3 (e.g. thirty passages in primary green monkey kidneycells, followed by three passages in fetal rhesus lung cells) and DENV-4PDK-48. These four candidate vaccine viruses were derived by tissueculture passage of wild-type parental DEN-1 16007, DEN-2 16681, DEN-316562 and DENV-4 1036 viruses, respectively. Any of these existing live,attenuated dengue viruses are contemplated of use in combination withthe DENV-4 chimeric virus constructs disclosed herein.

Previous human clinical trials with these attenuated viruses haveindicated that DEN-2 PDK-53 has the lowest infectious dose (50% minimalinfectious dose of 5 plaque forming units or PFU) in humans, is stronglyimmunogenic, and produces no apparent safety concerns. The DEN-1 PDK-13,DEN-3 PGMK-30/FRhL-3 and DENV-4 PDK-48 vaccine virus candidates havehigher 50% minimal infectious doses of 10,000, 3500, and 150 PFU,respectively, in humans.

DEN-2 PDK-53 virus vaccine candidate, henceforth abbreviated PDK-53, hasseveral measurable biological markers associated with attenuation,including temperature sensitivity, small plaque size, decreasedreplication in mosquito C6136 cell culture, decreased replication inintact mosquitoes, loss of neurovirulence for suckling mice anddecreased incidence of viremia in monkeys. Clinical trials of thecandidate PDK-53 vaccine have demonstrated its safety and immunogenicityin humans. Furthermore, the PDK-53 vaccine induces dengue virus-specificT-cell memory responses in human vaccine recipients.

In certain embodiments, a nucleic acid molecule can include a chimericflavivirus construct having a nucleic acid sequences encodingnonstructural proteins and at least one or more structural proteins froma live, attenuated dengue-2 virus and at least encoding one or morestructural proteins from a second flavivirus, wherein the chimericconstruct further comprises one or more mutations comprising a mutationin the envelope (E) protein at a position synonymous to amino acid 417,a mutation in the capsid protein at a position synonymous to position107, and a mutation in NS4A at a position synonymous to amino acidposition 17. In other embodiments, a nucleic acid can further include amutation in the envelope (E) protein at a position synonymous to aminoacid 417 that changes the wild type glutamic acid to a lysine. Yet othernucleic acid molecules disclosed herein can further include a mutationin the capsid (C) protein at a position synonymous to amino acid 107that changes a cysteine to a tyrosine. In other nucleic acid molecules,the mutation in the NS4A protein at a position synonymous to amino acid17 changes methionine (e.g. wild type sequence) to a leucine. It iscontemplated that the second flavivirus can be a DENV-1, DENV-3 orDENV-4. In certain embodiments, the nucleic acid molecule can include asecond flavivirus that is DENV-4. In other embodiments, the nucleic acidmolecules having a live attenuated dengue-2 backbone contains a mutationat position 57 in the 5′NCR, at position 53 of ns1 and position 250 ofns3. According to these embodiments, a live, attenuated dengue-2 viruscontains a mutation at position 53 of ns1 and position 250 of ns1 plus adengue-1, 3 or 4 substitution of one or more structural proteins. Incertain embodiments, a nucleic acid construct can be DENV-4e (SEQ IDNO:22), DENV-4h (SEQ ID NO:24) or DENV-4i (SEQ ID NO:9) capable ofinducing an immune response to dengue-4 virus in a subject. It iscontemplated herein that the structural sequences of dengue virusserotype 4 can be substituted using dengue-virus 1 or 3 and furthercontain the above referenced mutations for increasing an immune responseto the construct.

Some embodiments concern a nucleic acid molecule having a chimericflavivirus construct including a nucleic acid sequences encodingnonstructural proteins and at least one or more structural proteins froma live, attenuated dengue-2 virus and at least encoding one or morestructural proteins from a second flavivirus, wherein the attenuateddengue-2 contains a mutation at position 53 of ns1 and position 250 ofns3 but does not contain mutations in NS2A or NS4A. In certainembodiments, a nucleic acid construct can be DENV-4f (SEQ ID NO:30). Inother embodiments, nucleotide position 674 can be mutated to C from itswild-type nucleotide of G of DENV-4f. In yet other embodiments, amixture of DENV-2/DENV-4 constructs of the instant application can becombined in a pharmaceutically acceptable composition of use as animmunogenic agent against dengue virus infection.

In certain embodiments, an attenuated dengue-2 virus backbone ofDENV-2/DENV-4 constructs can further include one or moremutations/substitutions at positions 102-107 to a wild-type dengue 4sequence (e.g. 1086) in a DENV-2/DENV-4 construct. For example, one ormore of TITLLC at respective positions 102-107 from dengue-4 can replacewild type dengue-2 virus one or more of AGMIIM, at synonymous positions102-107, respectively. In accordance with these embodiments, aDEN-2/DEN-4 construct having a substitution in this region can furtherinclude a mutation of cysteine to an aromatic amino acid (e.g. tyrosine,tryptophan etc.).

In other embodiments, a DENV-2/DENV-4 construct of any immunogeniccompositions disclosed herein can be DENV-4g (SEQ ID NO:28) or DENV-4j(SEQ ID NO:32).

Immunogenic flavivirus chimeras having a dengue-2 virus backbone and atleast one structural protein of dengue-4 virus can be used for preparingthe dengue virus chimeras and methods for producing the dengue virus orflavivirus chimeras are described. The immunogenic flavivirus chimerasare provided, alone or in combination, in a pharmaceutically acceptablecarrier as immunogenic compositions to minimize, inhibit, or immunizeindividuals against infection by one or more dengue virus or flaviviralstrains, such as dengue virus serotypes DENV-4, alone or in combinationwith DEN-2, DEN-3 and DEN-1. When combined, the immunogenic flaviviruschimeras may be used as multivalent vaccines to confer simultaneousprotection against infection by more than one species or strain offlavivirus. In certain embodiments, the flavivirus chimeras are combinedin an immunogenic composition useful as a bivalent, trivalent ortetravalent vaccine against the known dengue virus serotypes or conferimmunity to other pathogenic flaviviruses by including nucleic acidsencoding one or more proteins from a different flavivirus. The nucleicacid sequence for each of the DEN-1, DEN-2, DEN-3 and DENV-4 viruses canbe used to generate a probe for use in detecting dengue virus in abiological sample in order, for example, to assess efficacy of thevaccine and/or level of a dengue virus infection.

In some embodiments, avirulent, immunogenic flavivirus chimeras providedherein contain the nonstructural protein genes of the attenuateddengue-2 virus (e.g. PDK-53), or the equivalent thereof, and one or moreof the structural protein genes or immunogenic portions thereof of theflavivirus against which immunogenicity is to be induced in a subject.For example, some embodiments concern a chimera having attenuateddengue-2 virus PDK-53 genome as the viral backbone, and one or morestructural protein genes encoding capsid, premembrane/membrane, orenvelope of the PDK-53 genome, or combinations thereof, replaced withone or more corresponding structural protein genes from DENV-4 virus orother flavivirus to be protected against, such as a different flavivirusor a different dengue virus serotype. In accordance with theseembodiments, the PDK-53 backbone is further mutated or reverted toincrease immunogenicity of the construct. Further, a nucleic acidchimera disclosed herein can have functional properties of theattenuated dengue-2 virus and is avirulent, but expresses antigenicepitopes of the structural gene products of DENV-4 in addition to otherflaviviruses and is immunogenic (e.g. induces an immune response to thegene products in a subject). The mutations and/or reversions do notaffect the attenuation and/or safety of the chimeric construct.

In another embodiment, a nucleic acid chimera can be a nucleic acidchimera having, but not limited to, a first nucleotide sequence encodingnonstructural proteins from an attenuated dengue-2 virus, and a secondnucleotide sequence encoding a structural protein from dengue-4 virusalone or in combination with another flavivirus. In other embodiments,the attenuated dengue-2 virus can be vaccine strain PDK-53 or 16681.Some embodiments include structural proteins of one or more of C, prM orE protein of a dengue or other flavivirus. Examples of flaviviruses fromwhich the structural protein may be selected include, but are notlimited to, DEN-1, DEN-2, DEN-3, West Nile virus, Japanese encephalitisvirus, St. Louis encephalitis virus, yellow fever virus and tick-borneencephalitis virus in combination with the DENV-4 constructs disclosedherein. In other embodiments, the structural protein may be selectedfrom non-flavivirus species that are closely related to theflaviviruses, such as hepatitis C virus.

Other aspects disclosed herein include that chimeric viruses can includenucleotide and amino acid substitutions, deletions or insertions forexample, in the DEN-2 PDK-53; these changes can reduce interference withimmunogenicity responses to DENV-4 virus. These modifications can bemade in structural and nonstructural proteins alone or in combinationwith the example modifications disclosed herein.

Embodiments herein include structural and nonstructural proteins of aflavivirus that can be any protein including or any gene encoding thesequence of the complete protein, an epitope of the protein, or anyfragment comprising, for example, five or more amino acid residuesthereof.

Certain embodiments disclosed herein provide for method for making thechimeric viruses of this invention using recombinant techniques, byinserting the required substitutions into the appropriate backbonegenome.

Flavivirus Chimeras

Dengue virus types 1-4 (DEN-1 to DENV-4) are mosquito-borne flaviviruspathogens. The flavivirus genome contains a 5′-noncoding region (5′-NC),followed by a capsid protein (C) encoding region, followed by apremembrane/membrane protein (prM) encoding region, followed by anenvelope protein (E) encoding region, followed by the region encodingthe nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and finallya 3′ noncoding region (3′NC) (See for example FIG. 1A). The viralstructural proteins are C, prM and E, and the nonstructural proteins areNS1-NS5. The structural and nonstructural proteins are translated as asingle polyprotein and processed by cellular and viral proteases.

Structure of Dengue Virus Genome

Flavivirus chimeras can be constructs formed by fusing non-structuralprotein genes from one type, or serotype, of dengue virus or virusspecies of the flaviviridae, with protein genes, for example, structuralprotein genes, from a different type, or serotype, of dengue virus orvirus species of the flaviviridae. Alternatively, a flavivirus chimeradisclosed herein is a construct formed by fusing non-structural proteingenes from one type, or serotype, of dengue virus or virus species ofthe flaviviridae, with further nucleotide sequences that direct thesynthesis of polypeptides or proteins selected from other dengue virusserotypes or other viruses of the flaviviridae.

In other embodiments, avirulent, immunogenic flavivirus chimerasprovided herein contain the nonstructural protein genes of theattenuated dengue-2 virus, or the equivalent thereof, and one or more ofthe structural protein genes, or antigenic portions thereof, of theflavivirus against which immunogenicity is to be conferred.

Other suitable flaviviruses for use in constructing the flaviviruschimeras can be wild-type, virulent DEN-1 16007, DEN-2 16681, DEN-316562 and DENV-4 1036 and attenuated, vaccine-strain DEN-1 PDK-13, DEN-2PDK-53, DEN-3 PMK-30/FRhL-3 and DENV-4 PDK-48. Genetic differencesbetween the DEN-1, DEN-2, DEN-3 and DENV-4 wild type/attenuated viruspairs are contemplated along with changes in the amino acid sequencesencoded by the viral genomes. Any DENV-4 strain of use herein wouldcontain synonymous mutations to the constructs contemplated and/ordisclosed herein.

Sequence listings for DEN-2 PDK-53 correspond to the DEN-2 PDK-53-Vvariant, wherein genome nucleotide position 5270 is mutated from an A toa T and amino acid position 1725 of the polyprotein or amino acidposition 250 of the NS3 protein contains a valine residue. The DEN-2PDK-53 variant without this nucleotide mutation, DEN-2 PDK-53-E, differsfrom PDK-53-V only in this one position. DEN-2 PDK-53-E has an A atnucleotide position 5270 and a glutamate at polyprotein amino acidposition 1725, NS3 protein amino acid position 250. It is understoodthat embodiments herein can include modified DEN-2 PDK-53 that includeone or more reversions/mutations of these positions to the nativederived sequence.

Sequence listings for DEN-3 16562 correspond to the variant whereingenome nucleotide position 1521 is a T and amino acid position 476 ofthe polyprotein or amino acid position 196 of the E protein contain aleucine. A second variant, present in DEN-3 16562 cultures has a T atnucleotide position 1521 and amino acid position 476 of the polyproteinor amino acid position 196 of the E protein contain a serine.

Sequence listings for DENV-4 PDK-48 correspond to the variant whereingenome nucleotide positions: 6957 is a T and amino acid position 2286 ofthe polyprotein and amino acid position 44 of NS4B protein is aphenylalanine, 7546 is a T and amino acid position 2366 of thepolyprotein and amino acid position 240 of NS4B protein is a valine, and7623 is a T and amino acid position 2508 of the polyprotein and aminoacid position 21 of NS5 protein is a tyrosine.

In certain embodiments, designations of the chimeras are based on theDEN-2 virus-specific infectious clone backbones and the structural genes(prM-E or C-prM-E) insert of other flaviviruses. DEN-2 for the dengue-2backbone, followed by the strain from which the structural genes areinserted. The particular backbone variant is reflected in next. Theparticular DEN-2 backbone variant from which the chimera was constructedis indicated by the following letter placed after a hyphen, parent 16681(P), PDK-53-E (E), or PDK-53-V (V); the last letter indicates theC-prM-E structural genes from the parental (P) strain or its vaccinederivative (V) or the prM-E structural genes from the parental (P) orits vaccine derivative (V1). For example; DEN-2/1-VP denotes the chimeracomprising the attenuated DEN-2 PDK-53V backbone comprising a valine atNS3-250 and the C-prM-E genes from wild-type DEN-1 16007; DEN-2/1-VVdenotes the DEN-2 PDK-53V backbone with the vaccine strain of dengue-1,DEN-1 PDK-13; DEN-2/1-VP1 denotes the DEN-2 PDK-53V backbone and theprM-E genes from wild-type DEN-1 16007; DEN-2/3-VP1 denotes the DEN-2PDK-53V backbone and the prM-E genes from wild-type DEN-3 16562;DEN-2/4VP1 denotes the DEN-2 PDK-53V backbone and the prM-E genes fromwild-type DENV-4 1036; and DEN-2/WN-PP1 denotes the DEN-2 16681 backboneand the prM-E genes from West Nile NY99. Other chimeras disclosed hereinare indicated by the same manner.

In one embodiment, chimeras disclosed herein contain attenuated DEN-2virus PDK-53 genome as the viral backbone, in which the structuralprotein genes encoding C, prM and E proteins of the PDK-53 genome, orcombinations thereof, are replaced with the corresponding structuralprotein genes from DENV-4 virus and optionally, another flavivirus to beprotected against, such as a different flavivirus or a different denguevirus strain. Newly discovered flaviviruses or flavivirus pathogens canalso be incorporated into the DEN-2 backbone.

In the nonstructural protein regions, a Gly-to-Asp (wild type-to-PDK-53)mutation was discovered at nonstructural protein NS1-53 (genomenucleotide position 2579); a Leu-to-Phe (wild type-to-PDK-53) mutationwas discovered at nonstructural protein NS2A-181 (genome nucleotideposition 4018); a Glu-to-Val (wild type-to-PDK-53) mutation wasdiscovered at nonstructural protein NS3-250 (genome nucleotide position5270); and a Gly-to-Ala mutation (wild type-to-PDK-53) was discovered atnonstructural protein NS4A-75 (genome nucleotide position 6599).

Attenuated PDK-53 virus strain has a mixed genotype at genome nt 5270. Asignificant portion (approximately 29%) of the virus population encodesthe non-mutated NS3-250-Glu that is present in the wild type DEN-2 16681virus rather than the NS3-250-Val mutation. As both genetic variants areavirulent, this mutation may not be necessary in an avirulent chimera.

Previously, it was discovered that avirulence of the attenuated PDK-53virus strain can be attributed to mutations in the nucleotide sequenceencoding nonstructural proteins and in the 5′ noncoding region. Forexample, a single mutation at NS1-53, a double mutation at NS1-53 and at5′NC-57, a double mutation at NS1-53 and at NS3-250 and a triplemutation at NS1-53, at 5′NC-57 and at NS3-250, result in attenuation ofthe DEN-2 virus. Therefore, the genome of any dengue-2 virus containingsuch non-conservative amino acid substitutions or nucleotidesubstitutions at these loci can be used as a base sequence for derivingthe modified PDK-53 viruses disclosed herein. Another mutation in thestem of the stem/loop structure in the 5′ noncoding region will provideadditional avirulent phenotype stability, if desired. Mutations to thisregion disrupt potential secondary structures important for viralreplication. A single mutation in this short (only 6 nucleotide residuesin length) stem structure in both DEN and Venezuelan equine encephalitisviruses disrupts the formation of the hairpin structure. Furthermutations in this stem structure decrease the possibility of reversionat this locus, while maintaining virus viability.

Mutations disclosed herein can be achieved by any method known in theart including, but not limited to, site-directed mutagenesis, directsynthesis, deletion, or other method using techniques known to thoseskilled in the art. It is understood by those skilled in the art thatthe virulence screening assays, as described herein and as are wellknown in the art, can be used to distinguish between virulent andavirulent backbone structures.

Construction of Flavivirus Chimeras

Flavivirus chimeras described herein can be produced by splicing one ormore of the structural protein genes of the flavivirus against whichimmunity is desired into a PDK-53 dengue virus genome backbone, or othermethods known in the art, using recombinant engineering to remove thecorresponding PDK-53 gene and replace it with a dengue-4 virus gene orother gene known in the art.

Alternatively, nucleic acid sequences of any construct disclosed herein,nucleic acid molecules encoding the flavivirus proteins, may besynthesized using any known nucleic acid synthesis techniques andinserted into an appropriate vector. Avirulent, immunogenic viruses ofembodiments herein can therefore be produced using recombinantengineering techniques known to those skilled in the art.

A target gene can be inserted into the backbone that encodes aflavivirus structural protein of interest for DENV-4, alone or incombination with another flavivirus. A flavivirus (e.g. dengue virus)gene to be inserted can be a gene encoding a C protein, a PrM proteinand/or an E protein. For example, a sequence inserted into the dengue-2backbone can encode both PrM and E structural proteins, or just a singlestructural protein. A sequence inserted into the dengue-2 backbone canencode all or one of C, prM and E structural proteins.

Suitable chimeric viruses or nucleic acid chimeras containing nucleotidesequences encoding structural proteins of other flaviviruses or denguevirus serotypes can be evaluated for usefulness as vaccines by screeningthem for phenotypic markers of attenuation that indicate avirulence andby screening them for immunogenicity. Antigenicity and immunogenicitycan be evaluated using in vitro and/or in vivo reactivity withflavivirus antibodies or immunoreactive serum using routine screeningprocedures known to those skilled in the art.

Dengue Virus Vaccines

In certain embodiments, chimeric viruses and nucleic acid chimeras canprovide live, attenuated viruses useful as immunogens or vaccines. Someembodiments include chimeras that exhibit high immunogenicity todengue-4 virus while producing no dangerous pathogenic or lethaleffects.

To reduce occurrence of DHF/DSS in subjects vaccinated against only oneserotype of dengue virus, a di-, tri or tetravalent vaccine is needed toprovide simultaneous immunity for two to all four serotypes of thevirus. A tetravalent vaccine can be produced by combining live,attenuated dengue-2 (e.g. dengue-2 PDK-53) with dengue-2/1, dengue-2/3,and dengue-2/4 novel chimeras described herein in a suitablepharmaceutical carrier for administration as a multivalent vaccineagainst all four dengue virus serotypes. Other formulations can includedivalent or trivalent formulations of the above where the formulationincludes one or more novel DENV-4 chimeric construct.

Chimeric viruses or nucleic acid chimeras disclosed in certainembodiments herein can include structural genes of either wild-type orattenuated viruses in a virulent or an attenuated DEN-2 virus backbone.For example, the chimera may express the structural protein genes ofwild-type DENV-4 1036 virus, and its candidate vaccine derivative ineither DEN-2 PDK-53 backgrounds. In certain embodiments, pharmaceuticalor experimental compositions disclosed herein can include one or moreconstructs having the designation of DENVax-4e, DENVax-4g, and/orDENVax-4h alone, or in combination with other flavivirus constructs. Incertain examples, these constructs can be used in combination with oneor more master virus seed (MVS) constructs disclosed herein (e.g.DEN-1/DEN-2). Other embodiments can include DENV-4 constructs disclosedherein in combination with other flavivirus chimeras such as those madeon a Yellow Fever backbone or West Nile backbone or other flavivirusbackbone where these flavivirus chimeras are capable of forming achimeric construct with a dengue virus serotype that when introduced toa subject induces an immune response to the virus in the subject.

Viruses used in the chimeras described herein can be grown usingtechniques known in the art. Virus plaque titrations are then performedand plaques counted in order to assess the viability and phenotypiccharacteristics of the growing cultures. Wild type viruses are passagedthrough cultured cell lines to derive attenuated candidate startingmaterials.

Chimeric infectious clones can be constructed from the various dengueserotype clones available. The cloning of virus-specific cDNA fragmentscan also be accomplished, if desired. The cDNA fragments containing thestructural protein or nonstructural protein genes can be amplified byreverse transcriptase-polymerase chain reaction (RT-PCR) from denguevirus RNA with various primers. Amplified fragments can be cloned intothe cleavage sites of other intermediate clones. Intermediate, chimericdengue virus clones can then be sequenced to verify the accuracy of theinserted dengue virus-specific cDNA.

In certain embodiments, full genome-length chimeric plasmids constructedby inserting the structural protein or nonstructural protein gene regionof dengue serotype viruses into vectors are obtainable using recombinanttechniques well known to those skilled in the art.

Nucleotide and Amino Acid Analysis

In certain embodiments, PDK-53, contains no amino acid mutations in theE protein relative to wild type dengue-2 virus; DEN-1, DEN-3 and DENV-4attenuated viruses can have amino acid mutations in the E protein.Wild-type DEN-3 16562 has been demonstrated to comprise traces of avariant comprising a T at nucleotide position 1521 which directsincorporation of a leucine at polyprotein position 476, amino acidresidue position 476 of the E protein. Each of the latter three virusescan possess a Glu-to-Lys (parent-to-vaccine) mutation in the E protein,although the mutation is located at a different amino acid residue inthe E protein. This substitution causes a shift from a negativelycharged amino acid to a positively charged one. The Glu-to-Lyssubstitution in the E protein of DENV-4 vaccine virus was the onlymutation present in the E protein, while the E proteins of DEN-1 andDEN-3 vaccine viruses had five and three amino acid mutations,respectively.

In certain embodiments, an NS1-53 mutation occurs in the DEN-2 PDK-53virus and is significant for the attenuated phenotype of this virus,because the NS1-53-Gly of the DEN-2 16681 virus is conserved in nearlyall flaviviruses, including the tick-borne viruses, sequenced to date.DENV-4 virus constructs disclosed herein can contain an amino acidmutation in the NS1 protein at position 253. This locus, which is aGln-to-His mutation in DENV-4 PDK-48 virus is Gln in all four wildserotypes of dengue virus. This Gln residue is unique to the dengueviruses within the flavivirus genus. The NS1 protein is a glycoproteinthat is secreted from flavivirus-infected cells. It is present on thesurface of the infected cell and NS1-specific antibodies are present inthe serum of virus-infected individuals. Protection of animals immunizedwith NS1 protein or passively with NS1-specific antibody has beenreported.

Certain mutations are found in NS2A, NS2B, NS4A, and NS4B proteins ofthe DEN-1, -2, -3 and -4 attenuated strains that are conservative innature. The NS4A-75 and NS4A-95 mutations of DEN-2 and DENV-4 vaccineviruses, respectively, occurred at sites of amino acid conservationamong dengue viruses, but not among flaviviruses in general.

Flaviviral NS3 protein possesses at least two recognized functions: theviral proteinase and RNA helicase/NTPase. The 698-aa long (DEN-2 virus)NS3 protein contains an amino-terminal serine protease domain(NS3-51-His, -75-Asp, -135-Ser catalytic triad) that is followed bysequence motifs for RNA helicase/NTPase functions (NS3-196-GAGKT),-284-DEAH, -459-GRIGR (SEQ ID NO:26), previously presented). None of themutations in the NS3 proteins of DEN-1, DEN-2, or DEN-3 virus occurwithin a recognized motif. NS3-510 Tyr-to-Phe mutation in DEN-1 PDK-13virus is a conservative mutation. Since the wild-type DEN-2, -3 and -4viruses contain Phe at this position, it is unlikely that the Tyr-to-Phemutation plays a role in the attenuation of DEN-1 virus. The NS3-182Glu-to-Lys mutation in DEN-1 PDK-13 virus occurred at a position that isconserved as Asp or Glu in most mosquito-borne flaviviruses and it mayplay some role in attenuation. This mutation was located 15 amino acidresidues upstream of the GAGKT (SEQ ID NO:27) helicase motif. In certaindengue-2 viruses, the NS3-250-Glu in DEN-2 16681 virus is conserved inall mosquito-borne flaviviruses except for yellow fever virus.

Nucleic acid probes of use in certain embodiments herein selectivelyhybridize with nucleic acid molecules encoding the DEN-1, DEN-3 andDENV-4 viruses or complementary sequences thereof. By “selective” or“selectively” is meant a sequence which does not hybridize with othernucleic acids to prevent adequate detection of the dengue virus.Therefore, in the design of hybridizing nucleic acids, selectivity willdepend upon the other components present in a sample. Hybridizingnucleic acids should have at least 70% complementarity with the segmentof the nucleic acid to which it hybridizes. As used herein to describenucleic acids, the term “selectively hybridizes” excludes the occasionalrandomly hybridizing nucleic acids, and thus, has the same meaning as“specifically hybridizing.” The selectively hybridizing nucleic acidsdisclosed herein can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%,and 99% complementarity with the segment of the sequence to which ithybridizes, preferably 85% or more.

Sequences, probes and primers which selectively hybridize to theencoding nucleic acid or the complementary, or opposite, strand of thenucleic acid are contemplated. Specific hybridization with nucleic acidcan occur with minor modifications or substitutions in the nucleic acid,so long as functional species-specific hybridization capability ismaintained. By “probe” is meant nucleic acid sequences that can be usedas probes or primers for selective hybridization with complementarynucleic acid sequences for their detection or amplification, whichprobes can vary in length from about 5 to 100 nucleotides, or preferablyfrom about 10 to 50 nucleotides, or most preferably about 18-24nucleotides.

If used as primers, the composition preferably includes at least twonucleic acid molecules which hybridize to different regions of thetarget molecule so as to amplify a desired region. Depending on thelength of the probe or primer, the target region can range between 70%complementary bases and full complementarity and still hybridize understringent conditions. For example, for the purpose of detecting thepresence of the dengue virus, the degree of complementarity between thehybridizing nucleic acid (probe or primer) and the sequence to which ithybridizes is at least enough to distinguish hybridization with anucleic acid from other organisms.

Nucleic acid sequences encoding the DENV-4, DEN-3 or DEN-1 virus can beinserted into a vector, such as a plasmid, and recombinantly expressedin a living organism to produce recombinant dengue virus peptides and/orpolypeptides.

Nucleic Acid Detection Methods

A rapid genetic test that is diagnostic for each of the vaccine virusesdescribed herein is provided by the current invention. This embodimentof the invention enhances analyses of viruses isolated from the serum ofvaccinated humans who developed a viremia, as well as enhancingcharacterization of viremia in nonhuman primates immunized with thecandidate vaccine viruses.

These sequences include a diagnostic TaqMan probe that serves to reportthe detection of the cDNA amplicon amplified from the viral genomic RNAtemplate by using a reverse-transciptase/polymerase chain reaction(RT/PCR), as well as the forward and reverse amplimers that are designedto amplify the cDNA amplicon, as described below. In certain instances,one of the amplimers has been designed to contain a vaccinevirus-specific mutation at the 3′-terminal end of the amplimer, whicheffectively makes the test even more specific for the vaccine strainbecause extension of the primer at the target site, and consequentlyamplification, will occur only if the viral RNA template contains thatspecific mutation.

Automated PCR-based nucleic acid sequence detection system can be used,which is becoming widely used in diagnostic laboratories. The TaqManassay is a highly specific and sensitive assay that permits automated,real time visualization and quantitation of PCR-generated amplicons froma sample nucleic acid template. TaqMan can determine the presence orabsence of a specific sequence. In this assay, a forward and a reverseprimer are designed to anneal upstream and downstream of the targetmutation site, respectively. A specific detector probe, which isdesigned to have a melting temperature of about 10° C., higher thaneither of the amplimers and containing the vaccine virus-specificnucleotide mutation or its complement (depending on the strand of RT/PCRamplicon that is being detected), constitutes the third primer componentof this assay. A probe designed to specifically detect a mutated locusin one of the chimeric constructs can contain a specific nucleotidechange for detecting any mutation.

One strategy for diagnostic genetic testing makes use of molecularbeacons. The molecular beacon strategy also utilizes primers for RT/PCRamplification of amplicons, and detection of a specific sequence withinthe amplicon by a probe containing reporter and quencher dyes at theprobe termini. In this assay, the probe forms a stem-loop structure. The5′- and 3′-terminal reporter dye and quencher dye, respectively, arelocated at the termini of the short stem structure, which brings thequencher dye in close juxtaposition with the reporter dye. Thestem-structure is melted during the denaturation step of the RT/PCRassay. If the target viral RNA contains the target sequence and isamplified by the forward and reverse amplimers, the opened loop of theprobed hybridizes to the target sequence during the annealing step ofthe cycle. When the probe is annealed to either strand of the amplicontemplate, the quencher and reporter dyes are separated, and thefluorescence of the reporter dye is detected. This is a real-timeidentification and quantitation assay that is very similar to the TaqManassay. The molecular beacons assay employs quencher and reporter dyesthat differ from those used in the TaqMan assay.

Pharmaceutical Formulations

Any pharmaceutical formulation known in the as for a vaccine iscontemplated herein. In certain embodiments, a formulation can contain,DENV-4 constructs alone or one or more additional DEN serotype (or otherflavivirus compositions) in various ratios in combination with DENV-4constructs disclosed herein, depending on predetermined exposure to orexistence of dengue virus subtype prevalence in a region. It iscontemplated that formulations can contain other agents of use invaccination of a subject including, but not limited to other active orinactive ingredients or compositions known to one skilled in the art. Incertain embodiments, an adjuvant may be included in a formutationdisclosed herein.

Other aspects of the present invention can include modulating an immuneresponse to a vaccine against dengue virus to a subject. Vaccinesagainst dengue virus may include a composition comprising ratios ofserotypes of dengue virus, live attenuated dengue virus, or fragmentsthereof such as proteins or nucleic acids derived or obtained fromdengue virus serotypes. Ratios of various serotypes may be equal orcertain serotypes may be represented more than others depending on needor exposure or potential exposure to the virus. In accordance with theseembodiments, a ratio may be a 1:2, 1:3, 1:4, 1:10, 1:20; 1:1:1, 1:2:2,1:2:1, 1:1:1:1, 1:2:1:2; 1:3:1:3, 2:3:3:3, 5:4:5:5, 4:4:4:5, 1:2:2,4:4:5:5, 4:4:5:6 or any ratio for any of serotypes 1, 2, 3 incombination with the DENV-4 constructs disclosed herein, depending onfor example, number of serotypes represented in the formulation,predetermined response and effect desired. The last number representsthe amount of DENV-4 construct in a formulation. Each number representsthe power of ten (6=10⁶ PFU). It is contemplated that any dengue virusserotype formulation may be used to generate a vaccine (e.g. attenuatedvirus etc.) of use in administration to a subject in need thereof.

Embodiments herein provide for administration of compositions tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo. By “biologically compatible form suitable foradministration in vivo” is meant a form of the active agent (e.g.pharmaceutical protein, peptide, or gene etc. of the embodiments) to beadministered in which any toxic effects are outweighed by thetherapeutic effects of the active agent. Administration of atherapeutically active amount of the therapeutic compositions is definedas an amount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof a compound may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of antibody toelicit a desired response in the individual. Dosage regima may beadjusted to provide the optimum therapeutic response.

In one embodiment, the compound (e.g. pharmaceutical protein, peptideetc. of the embodiments) may be administered in a convenient manner suchas subcutaneous, intravenous, intradermal, by oral administration,inhalation, transdermal application, intradermal, intravaginalapplication, topical application, intranasal or rectal administration.Depending on the route of administration, the active compound may becoated in a material to protect the compound from degradation byenzymes, acids and other natural conditions that may inactivate thecompound. In one embodiment, the compound may be administeredintranasally, such as inhalation.

A compound may be administered to a subject in an appropriate carrier ordiluent, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. The term “pharmaceutically acceptablecarrier” as used herein is intended to include diluents such as salineand aqueous buffer solutions. It may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. The active agent may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use may beadministered by means known in the art. For example, sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion may be used. In all cases, the composition can be sterile andcan be fluid to the extent that easy syringability exists. It might bestable under the conditions of manufacture and storage and may bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The pharmaceutically acceptable carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention ofmicroorganisms can be achieved by heating, exposing the agent todetergent, irradiation or adding various antibacterial or antifungalagents.

Sterile injectable solutions can be prepared by incorporating activecompound in the required amount with one or a combination of ingredientsenumerated above, as required.

Aqueous compositions can include an effective amount of a therapeuticcompound, peptide, epitopic core region, stimulator, inhibitor, and thelike, dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. Compounds and biological materials disclosed herein canbe purified by means known in the art.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above.It is contemplated that slow release capsules, timed-releasemicroparticles, and the like can also be employed. These particularaqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

The active therapeutic agents may be formulated within a mixture tocomprise about 10² to about 5×10⁶ PFU of each construct contemplatedherein. Single dose or multiple doses can also be administered on anappropriate schedule for a predetermined condition. In certainembodiments, a dual dose on day 0 may be administered in singleanatomical or multiple anatomical locations in order to induce an immuneresponse with reduced interference or different lymph nodes. In certainembodiments, a mono-, bi-, tri- or tetravalent formulation of denguevirus constructs may be administered to a subject. Any of theseformulations can be provided to a subject as a single or in multipledoses. In certain embodiments, one dose can be administered then a boostsome time later may be provided.

In another embodiment, nasal solutions or sprays, aerosols or inhalantsmay be used to deliver the compound of interest. Additional formulationsthat are suitable for other modes of administration includesuppositories and pessaries. A rectal pessary or suppository may also beused. In general, for suppositories, traditional binders and carriersmay include, for example, polyalkylene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1% 2%.

The pharmaceutical compositions containing the α1-antitrypsin, analogthereof, or inhibitor of serine protease activity or a functionalderivative thereof may be administered to individuals, particularlyhumans, for example by subcutaneously, intramuscularly, intranasally,orally, topically, transdermally, parenterally, gastrointestinally,transbronchially and transalveolarly.

In certain embodiments of the methods of the present invention, thesubject may be a mammal such as a human or a veterinary and/or adomesticated animal.

In one embodiment of the present invention, methods provide forvaccinating a subject preparing to travel to a country with denguevirus. In other embodiments, a subject may be a resident in an endemicarea. It is contemplated that a subject may be administered a singleinjection or dual injections on day 0, optionally followed by a boostless than 30 days, 2 months, 3 months, 6 months or as much as one yearlater.

Kits

Other embodiments concern kits of use with the methods (e.g. methods ofapplication or administration of a vaccine) and compositions describedherein. Some embodiments concern kits having vaccine compositions of useto prevent or treat subjects having, exposed or suspected of beingexposed to one or more dengue viruses. In certain embodiments, a kit maycontain one or more than one formulation of dengue virus serotype(s)(e.g. attenuated vaccines) at predetermined ratios. Kits can beportable, for example, able to be transported and used in remote areassuch as military installations or remote villages. Other kits may be ofuse in a health facility to treat a subject having been exposed to oneor more dengue viruses or suspected of being at risk of exposure todengue virus.

Kits can also include a suitable container, for example, vials, tubes,mini- or microfuge tubes, test tube, flask, bottle, syringe or othercontainer. Where an additional component or agent is provided, the kitcan contain one or more additional containers into which this agent orcomponent may be placed. Kits herein will also typically include a meansfor containing the agent, composition and any other reagent containersin close confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained. Optionally, one or more additional agents such asimmunogenic agents or other anti-viral agents, anti-fungal orantibacterial agents may be needed for compositions described, forexample, for compositions of use as a vaccine against one or moreadditional microorganisms.

Embodiments of the present invention are further illustrated by thefollowing non-limiting examples, which are not to be construed in anyway as imposing limitations upon the scope thereof. It should beappreciated by those of skill in the art that the techniques disclosedin the Examples which follow represent techniques discovered to functionwell in the practices disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope herein.

EXAMPLES Example 1

In certain exemplary methods, DENV-4 chimera constructs are generated ofuse in pharmaceutically acceptable compositions.

FIG. 1A illustrates an exemplary viral backbone of some embodimentsdisclosed herein referred to as DEN-2 PDK-53 genome (previouslydisclosed in PCT/US01/05142, U.S. application Ser. No. 10/204,252, eachincorporated herein by reference in their entirety for all purposes)which can be used to generate modified DENV-4 constructs (also indicatedas a second generation). As shown in FIG. 1A, specific amino acidsubstitution mutations in the nonstructural regions, such as NS1-G53D,NS3-E250V and a nucleotide substitution mutation in the 5′ non-codingregion, C57T in the DEN-2 PDK-53 genome have been identified. OriginalDENV-1, 3, and 4 constructs (also indicated as first generation) weregenerated by replacing the coding sequence of prM and E in the DENV-2backbone with structural coding sequences of respective serotypes (FIG.1B). To generate modified DENVax-4 constructs that can boost replicationefficiency in vitro and in vivo, and improve immunogenicity in the host,point mutations were introduced to the non-coding region, structuralprotein coding sequences and/or non-structural regions of the originalDENVax-4 construct. For example, modifications can be made to the aminoacid sequence upstream of the current DENVax-4 C/prM cleavage site tomimic wild-type dengue-4 as opposed to dengue-2 (which DENVax-4currently contains).

Some additional modifications in certain exemplary DENV-4 constructs areprovided in Table 1. Illustrated below are sequences included in certainmodified DENVax-4 constructs (DENVax-4b, DENVax-4c, and DENVax-4d)aligned to the wild type sequence of DENV-2, DENV-1 and originalDENVax-4. The selected changes in these sequences are in bold andunderlined:

       C-100      Capsid ---  prM DENV-2: NILNRRRRSAGMIIMLIPTVMA FHLTTRNSEQ ID NO: 1 DENV-4 WT: NILN G R K RS TITLLC LIPTVMA FHL S TR DSEQ ID NO: 2 DENVax-4ori: NILNRRR S SAGMIIMLIPTVMA FHLTTR D SEQ ID NO: 3DENVax-4b: NILNRRR S S TITLLC LIPTVMA FHL S TR D SEQ ID NO: 4 DENVax-4c:NILN G R K RS TITLLC LIPTVMA FHL S TR D SEQ ID NO: 5 DENVax-4d: *ILN G RK RS TITLLC LIPTVMA FHL S TR D SEQ ID NO: 6

FIG. 4 presents a schematic map of the original and modified DENV-4constructs. DENVax-4e is generated using DENVax-4b sequence as abackbone, and further incorporates a C to Y mutation at amino acid 107of Capsid protein. DENVax4f, DENVax4g and DENVax4j were built on theDENVax-4a backbone, but contain wild type reversions of the attenuatingmutations of the PDK-53 sequence as indicated in FIG. 4: DENVax-4fcontains wild type (DEN-2 16681) NS2A and NS4A; DENVax-4g contains wildtype 5′NCR, NS2A and NS4A; and DENVax-4j contains wild type 5′NCR.DENVax4h has an E to K substitution at Envelope 417 in the DENV4 1036sequence. DENVax4i has a methionine to leucine amino acid substitutionat NS4A position 17 (synonymous position 6424 A to T of the genomicnucleic acid or amino acid position 2110 of the construct). DENVax4k hasthe PrM/E genes from DENV-4 H241 strain instead of 1036 strain (Table1). Mutations constructed in DENVax4e, h, and i were based on sequencedata from DENVax4 and DENVax-4b serial passages in Vero cells.

DENVax-4b contains 7 total amino acid changes, DENVax-4c contains 9total amino acid changes, and DENVax-4d contains an amino acid deletionresulting in a frame shift of the sequence. RNA was transcribed andelectroporated into Vero cells for amplification and virus rescue.DENVax-4b and DENVax-4c were tested for growth efficiency in Vero andC6/36 cells in a growth kinetics experiment, with DENVax4-P2, DENVax4-P8and DENVax2-P2 used as controls. DENVax-4-P2 and DENVax-2-P2 are virussamples that have not been genotypically selected by viral plaquepurification, a process in which individual viral plaques are pickedfrom a DENVax-infected Vero cell monolayer, and then over-layed withagarose gel containing neutral red to visualize single plaques.DENVax-4-P8 had been selected by plaque purification to obtain a virusstock with a clonal DENVax-4 genotype. This procedure is done togenerate a master seed virus (MVS) with no reverted attenuatingmutations. DENVax-4c did not reach an adequate peak titer after growthin Vero cells, and had a slower initial growth rate than either DENVax-4or DENVax-4b. There was no significant difference between the peaktiters of DENVax-4 and DENVax-4b in the Vero growth curve, and both hadsimilar initial growth rates. In the analysis C6/36 mosquito cells,growth of DENVax-4b and DENVax-4c both reached peak titers that weresignificantly less than wild type DENV-4, confirming their attenuation.

TABLE 1 Exemplary DENV-4 chimeric constructs: Differences in theconstructs compared to DENV-4/DEN-2 PDK-53 previously disclosedExemplary DENV- 4 Constructs Modifications DENVax-4e Capsid 107 C to Ymutation at position 416 with G mutated to A of DENVax-4b backboneDENVax-4f Attenuating mutations in NS2 and NS4 reverted to correspondingsequences of Dengue virus strain 16681 DENVax-4g Attenuating mutationsin 5′ NCR, NS2 and NS4 reverted to corresponding sequences of Denguevirus serotype 2 strain 16681 DENVax-4h Envelope 417 E to K mutation atposition 2185 with G mutated to A of DENVax-4b backbone DENVax-4i NS4A17 M to L mutation at position 6424 with A mutated to T of DENVax-4bbackbone DENVax-4j Attenuating mutations in 5′ NCR reverted tocorresponding sequences of Dengue virus serotype 2 strain 16681DENVax-4k Coding sequence of prM and E of DENV-2 backbone replaced withstructural coding sequences of Dengue virus serotype 4 strain H241

Example 2 Generation of Full-Length Infectious cDNA Clones

To generate full-length infectious cDNA clones containing modifiedDENVax-4 construct sequences, a multi-step digestion/ligation scheme canbe used. It can have the following steps: 1) insert the AgeI/MluIsynthetic fragment including a modified nucleic acid sequence or amodified structural protein coding sequence (for example, b, c or d, orany of the above constructs) into pD2/3-PP1-5′ to generate pD2/4i-b, cor d; 2) digest the pDENVax-4 full length cDNA clone to extract theMluI/NgoMIV fragment and insert it into the corresponding position ofpD2/4i-b, c or d to obtain pD2/4i-b, c or d; 3) digest the pDENVax-4full length cDNA clone to extract NgoMIV/XbaI fragment and insert itinto the corresponding position of pD2/4i-b, c or d to generate the fulllength infectious clones containing modified sequences. The sequences ofthe final full-length infectious clones were confirmed by sequenceanalysis.

Virus Generation

The cDNA clones for each of the modified construct was transcribed intogenomic viral RNA. The RNA was transformed into Vero cells byelectroporation. The viruses were grown for 12 days while monitoringCPE, and harvested. This first harvest after electroporation was termedP1 (Passage 1). The subsequent amplifications and passages were calledP2, P3, etc. There was limited CPE for the original DENVax-4, andDENVax-4b, and c, whereas DENVax-4d did not generate any noticeable CPE.Unlike other viruses when grown in vitro, the dengue viruses do notproduce much CPE when/grown in Vero cells. Although the DENVax-4d virusdid not generate any CPE in vitro, it was amplified in parallel with theother strains. Amplified P1 viruses (for example, DENVax-4b, c, d P1)produced high enough titers to perform sequence analysis and growthcurve experiments. DENVax-4d had no titer and therefore, no virus wasmade after electroporation.

The DENVax-4b and -4c viruses were fully sequenced. The DENVax-4b-P2virus had two mutations. Nucleotide 416 was at the position of amodification engineered in the capsid (near the C/prM junction) of theDENVax-4b virus. Since this was a mixed population, the nt 416 wasreverting back to the “A” nucleotide instead of the engineered “G”nucleotide, causing the expected amino acid arginine to instead be alysine. The second mutation found in the DENVax-4b-P2 virus was atnucleotide 8769. This caused a change in the amino acid from theexpected glutamine to a proline. This mutation was in the NS5 generegion of the infectious clone. The DENVax-4c-P2 virus had fourmutations. They were all complete conversions, unlike the DENVax-4bvirus that had mixed populations. The mutation at nucleotide 400 in thecapsid region of the genome affected an engineered modification at theC/prM junction. This caused the expected amino acid at that position tobe a proline instead of threonine. The other three mutations were in thenonstructural genes, two of which caused amino acid changes, and onethat was a silent mutation.

Phenotypic and Genetic Characterization of the Viruses

Phenotypic characterization of the viruses was performed in Vero cellsand C6/36 (Aedes albopictus) cells. FIGS. 5-6 represent exemplaryphenotypic character of the virus variants generated from the modifiedDENVax-4 constructs. Plaque size is an important attenuation marker, andwas analyzed for each of the virus variants. As shown in FIG. 5, theplaque size of the DENVax-4b viruses was roughly 0.3 cm in diameter. Theplaque size of the DENVax-4c viruses was about 0.1 cm in diameter. Theplaque size of the DENVax-4h viruses was about 0.3 cm in diameter. Theplaque size of the DENVax-4i viruses was about 0.1 cm in diameter. Theplaque size of the DENVax-4c viruses was about 0.2 cm in diameter. Eachof the sizes represents an average of 5-10 plaques. The plaques in the band c candidate viruses, especially DENVax-4c were not homogenous. Therewas a mixed population of viruses with some small and some largeplaques. The sequence variability of these small and large plaques canbe characterized for possible nucleotide changes which would contributeto attenuation and fitness in vitro. If needed, these mutations could beengineered into further DENVax-4 variants. FIGS. 6A-6F present exemplaryresults of ImmunoFoci of the wild type DENV-4 virus (A) and each of theDENVax-4b-f virus variants (B-F).

In some exemplary methods, growth curves of DENV-4 constructs generatedviruses were determined. Monolayers of Vero cells were infected at a MOIof 0.001 with various DENV-4 construct compositions (for example,DENVax-4b-j, and DENVax-4P1). In some exemplary experiments, sampleswere taken every other day through day 13, and aliquots were titrated.The DENVax-2-P2 virus reached peak titers more rapidly. The DENVax-4bvirus was similar in peak titer and growth rate to the DENVax-4-P2 andP8 viruses. The DENVax-4c virus initially had a slower growth rate, butit finally reached a similar peak titer. The efficiency and peak titersof the DENVax-4b and DENVax-4c viruses were comparable to the originalDENVax-4. In other exemplary experiments, samples were obtained everyother day from day 2 through day 12. Harvested media was retained andstabilized at days 7, 9 and 11 for further study. Samples from day 2 to12 were titered by IFA. The DENVax-4e-4h viruses showed similar peaktiter to control DENVax-4 (FIG. 7).

To demonstrate attenuation, the replication efficiency of each vaccinevirus should be decreased in C6/36 mosquito cells as compared to thewild-type virus. This phenotype is an essential safety feature of DENVaxvaccine viruses, to ensure that there is no potential for transmissionof the attenuated chimeric viruses in nature. In some exemplary methods,growth in C6/36 mosquito cells was conducted to compare the growthcharacteristics of the DENVax4b and DENVax-4c viruses to the wild-typedengue 4 virus (strain 1036). In other exemplary experiments, comparisonwas done between DENVax4e-4j and original DENVax4. Duplicate flasks ofC6/36 cells were infected at a MOI of 0.001 with each of the P2 viruses(-b, -c, and wild-type) and grown for 14 days. The dengue 4 wild-typevirus (WT D4 1036) replicated most efficiently and to the highest titer.The DENVax-4b virus replicated reasonably well in the C6/36 cells,reaching a peak titer of 2.7×10⁶ pfu/mL by day 14. DENVax4c virus wasvery slow growing until after day 6 when growth was accelerated untilday 14, and reaching a peak titer of 2.2×10⁴ pfu/mL. At day 6, bothDENVax-4b and DENVax4c were similarly attenuated for growth as comparedto the wild-type in C6/36 cells, and had similar titers to the originalDENVax-4. In other exemplary experiments, chimeric dengue viruses ofcertain embodiments herein were grown in C6/36 mosquito cells. Growth inthe mosquito cells DENVax4e-4j was compared to a control DENVax4.Duplicate flasks of C6/36 cells were infected at a MOI of 0.001 witheach of the P2 viruses and grown for 12 days. Samples were harvested onday 2 through day 12. Growth in the mosquito cells were compared togrowth in Vero cell. Culture media was obtained and stabilized at days7, 9 and 11. Samples from day 2 to 12 were titered by IFA and analyzedfor virus production. Virus titer and growth of the constructs werecompared to control DENVax4 (see FIG. 8).

FIGS. 7-8 represent exemplary graphs illustrating growth curves ofviruses of various DENV4 constructs in Vero cells (FIG. 7) and in C6/36mosquito cells (FIG. 8). Several novel DENVax4 constructs of certainembodiments herein produced higher titers in Vero cells (FIG. 7) thanthe control DENVax4 and lower titers in mosquito cells (FIG. 8) thancontrol DENVax4, as desired for further assessment in animal models (seeTable 2 below).

Safety and Efficacy of the Virus Variants in Mice

Studies were performed to analyze the immunogenicity of DENVax-4b, c orother variant viruses. Table 2 presents an exemplary study design.Groups of 10 AG129 mice were vaccinated intradermally (in the footpad)with 10⁴ PFU each of the original DENVax-4, and the separate variants. Acontrol group was injected with excipient solution only. These micereceived a booster dose after 42 days. Serum samples for serology weretaken on days 42 and 56 post primary inoculation, and seroconversion wasanalyzed by PRNT. There was no significant improvement in theimmunogenicity of DENVax-4 when using the second generation DENVax-4band c variants as immunogens.

TABLE 2 Summary of Mouse Study Design Number of Number of Group Doseimmunizations animals 1 DENVax-4 10⁴ PFU 2 10 2 DENVax-4b 10⁴ PFU 2 10 3DENVax-4c 10⁴ PFU 2 10 4 TFA NA 2 10

To further modify and amplify the immunogenicity of DENVax-4b, thisvirus was adapted in vitro to Vero cell growth. This was performed by 10blind passages in Vero cells, with the hypothesis that adaptation inmammalian cell culture would boost the replicative capacity of the virusand thus its immunogenicity. This “new” second generation DENVax-4 wastermed DENVax-4b-P10 (Passage-10).

Another study was performed to evaluate different tetravalentformulations of dengue virus vaccine including three separate DEN4viruses; first generation, second generation (4b construct) and thehomologous wildtype DEN4 1036. Groups of 6 mice were injectedintradermally with a tetravalent vaccine formulation containing 10⁴ PFUDENVax-1, 10³ PFU DENVax-2, 10⁴ PFU DENVax-3 and 10⁵ PFU DENVax 4(4:3:4:5) in a 50 μL volume. Control mice were injected by the sameroute with 10⁵ PFU of 1^(st) generation, 2^(nd) generation or wildtype1036 monovalent DENV4. A group of mice injected with diluent only wasincluded as a control. All mice received a booster injection on day 42with the corresponding vaccine formulation, and mice were bled on days0, 21, 41 and 56 post primary inoculation to assess the presence ofneutralizing antibodies against all four DENV serotypes. On day 56, micefrom groups 4, 5 and 6 were challenged with DENV-2 (NGC strain) toassess for survival (there is currently no mouse-adapted lethal DENV4strain to use for efficacy analysis). As illustrated in Table 3, thefirst and second generation DENVax-4 vaccines have comparableimmunogenic profile when administered alone. However, when they aregiven in the context of tetravalent dengue virus vaccine, immuneresponses to DENV-4 are diminished due to interference affecting onlythe second generation DENVax-4. Wild type DENV4 was highly immunogenicand in the context of tetravalent dengue virus vaccine it interferes andsuppresses the neutralizing antibody responses elicited by the otherthree dengue virus vaccines. Wild type DENV4, the first generation andthe second generation DENVax-4 provided partial protection againstheterologous DENV-2 challenge.

Safety and Efficacy of Second Generation DENVax in NHP

TABLE 3 Mouse study #2 study design and PRNT results Group Dose DENV1DENV2 DENV3 DENV4 1 DENVax (1^(ST) 4:3:4:5 1280 640 >1280 640 generationDENVax-4) (320) (320) (320) (160) 2 DENVax (DENVax-4 4:3:4:5 160 160 320640 was replaced with wild (320) (160) (160) (640) type DENV-4) 3 DENVax(DENVax-4 4:3:4:5 >1280 640 >1280 80 was replaced with 2^(nd) (640)(320) (640)  (80) generation DENVax-4) 4 DENVax-4 (2^(nd) 10{circumflexover ( )}5 X X X 320 generation)  (80) 5 Wild type DENV-4 10{circumflexover ( )}5 X X X 640 (320) 6 DENVax-4 (1^(st) 10{circumflex over ( )}5 XX X 320 generation)  (80)

The immunogenicity of DENVax in groups of four Cynomologus macaques wasevaluated after subcutaneous injection of each tetravalent DENVaxformulation (Table 4 and FIG. 2) in 0.5 mL injection volume. The goal ofthis study was to evaluate the lead second generation DENVax-4construct, as compared with the first generation DENVax-4. Additionally,different methods were explored to improve the neutralizing antibodyresponses to DENV-4 elicited by DENVax. These included testing aformulation which contained 10× the dose of DENVax-4 as used previously(termed “new formulation”), and also lowering the DENVax-2 component ofthe tetravalent mixture, as this vaccine is the most immunogenic of thefour vaccine virus strains. Different dosing days were tested, andpriming and boosting on the same day were compared (Day 0) to priming onDay 0 and boosting on Day 60. Samples for serology were taken on days 0,28, 58, 73, and 90. The kinetics of neutralizing antibody titers againstDENV-4 variant virus and the original was shown in FIG. 3 as an example,where comparable levels were observed.

TABLE 4 NHP Study Design: Groups Vaccine Source Formulation Treatment 1DENVax Formulation 2e4, 5e4, 1e5, 3e5 2 dose (day 0) (Clinical Stock) 2DENVax Formulation 2e4, 5e4, 1e5, 3e5(4b) 2 dose (day 0) (4b-P10) 3DENVax Formulation 2e4, 5e4, 1e5, 3e6 2 dose (day 0) (Clinical Stock)w/10x DENVax-4 4 DENVax Formulation 2e4, 5e4, 1e5, 3e6 2 dose (0, 60)(Clinical Stock) w/10x DENVax-4 5 DENVax New 2e4, 1e4, 1e5, 3e6 2 dose(0, 60) formulation

Vaccine formulations for the NHP studies were prepared in bulk. Group 1received vaccine which was cGMP manufactured and is identical to thevaccine which is currently being tested in human clinical trials.Vaccines were given to the NHPs and subsequently back-titrated todetermine the actual dose. These results are presented in Table 5 below.

TABLE 5 Vaccine Stocks-back titration results: desired desired iFFU/dosedose desired dose back dose back DENVax GP1 GP2 back titration GP3 and 4titration GP5 titration D1 2.00E+04 2.00E+04 1.90E+04 2.00E+04 1.80E+042.00E+04 1.80E+04 D2 5.00E+04 5.00E+04 2.60E+04 5.00E+04 2.40E+041.00E+04 3.60E+03 D3 1.00E+05 1.00E+05 1.80E+05 1.00E+05 1.40E+051.00E+05 1.70E+05 D4 3.00E+05 300000 2.10E+05 3.00E+06 2.90E+06 3.00E+063.00E+06 (4b)

Samples for viremia were taken after the primary dose on days −11(baseline), 3, 5, 7, 9, 11, 13, 15, 17, 21, 28, 58, 62 and 66. RNA wasextracted from the serum sample and virus titer was determined bytetraplex qRT-PCR assay. The only virus which gave any detectableviremia was DENVax-2, and this resolved by day 21 after primaryvaccination. No virus was detected after the booster dose of vaccine wasadministered.

Serology was evaluated using a high throughput PRNT assay in a 96 wellplate. Comparison of the first and second generation DENVax-4 (Group 1compared to group 2) showed no significant difference in immunogenicityof these two viruses (Table 6).

TABLE 6 Geometric Mean Titers of NHP study. Group DEN d0 d28 d58 d73 d90d128 d149 d181 d210 1 1 5 226.3 134.5 160.0 95.1 67.3 95.1 226.3 226.35455a 2 5 1810.2 1280.0 905.1 905.1 761.1 761.1 761.1 380.5 0.0 3 5226.3 190.3 320.0 226.3 95.1 134.5 226.3 160.0 4 5 28.3 20.0 134.5 56.623.8 33.6 113.1 67.3 2 1 5 33.6 16.8 47.6 40.0 23.8 23.8 20.0 67.3 5455b2 5 1076.3 1280.0 905.1 1076.3 1076.3 1280.0 640.0 640.0 0.0 3 5 67.333.6 226.3 80.0 23.8 28.3 28.3 33.6 4 5 23.8 11.9 80.0 47.6 23.8 33.623.8 20.0 3 1 5 80.0 47.6 56.6 67.3 40.0 33.6 134.5 134.5 5456 2 5 380.5320.0 538.2 538.2 190.3 508.0 269.1 269.1 0.0 3 5 95.1 80.0 134.5 95.140.0 40.0 160.0 113.1 4 5 95.1 134.5 160.0 160.0 134.5 134.5 320.0 226.34 1 5 16.8 16.8 80.0 56.6 23.8 23.8 16.8 20.0 5456 2 5 134.5 134.5 269.1320.0 134.5 134.5 95.1 80.0 0.60 3 5 23.8 23.8 134.5 95.1 23.8 20.0 28.328.3 4 5 23.8 20.0 134.5 113.1 56.6 80.0 28.3 28.3 5 1 5 20.0 11.9 134.5134.5 33.6 20.0 28.3 14.1 5356 2 5 28.3 8.4 95.1 80.0 10.0 5.9 5.0 5.00.60 3 5 23.8 11.9 190.3 190.3 23.8 28.3 33.6 28.3 4 5 33.6 33.6 269.1134.5 113.1 56.6 80.0 67.3

Evaluating a higher dose of DENVax-4 (Group 3) revealed that asignificant difference in the kinetics of DENV-4 neutralizing antibodyresponses could be obtained. The peak titers remained roughly equivalent(within a 2-fold dilution range) when comparing the two differentvaccine formulations, but the rate in which the peak titer to DENV-4 wasobtained was much earlier when the immunization was performed with agreater amount of DENVax-4. Further, when the amount of DENVax-2 waslowered in the formulation (Group 5), this had no marked difference inthe kinetics or peak titer of DENV-4 in serum responses in thistetravalent DENVax vaccine.

FIG. 9 represents a flow chart of one exemplary procedure for testingsafety and efficacy of the virus construct disclosed herein in an animalmodel. For example, composition(s) that include one or more chimericDengue viruses of use as a vaccine, such as compositions of DENVax4e,DENVax4f or DENVax4h, or other chimeric constructs or a controlcomposition can be tested in animal models, e.g. AG129. Animals can beprimed on day 0, and subsequently boosted with the same or differentDengue virus vaccine compositions on or before day 30 or day 42 or otherappropriate time. Then, the animals can be challenged with exposure toone or more Dengue virus serotypes to assess induction of an immuneresponse to the challenge. Blood samples can be collected, for example,on days 0, 30, 41, 48, 52 or 84 or other appropriate day to test forneutralizing antibodies, and on other days such as 5 and 47 to test forviremia (or as deemed appropriate for the composition and administrationprotocol chosen).

Virus Cloning and Rescue to P1

Plasmid mutagenesis was used to create the new DENV-4 chimeric clones ofuse in vaccine compositions disclosed herein. Primers coding for pointmutations were synthesized and used to amplify an entire new infectiousclone. The template was digested by DpnI and the subsequent plasmid wassequenced. RNA was transcribed and electroporated into Vero cells tocreate a virus at passage level 0 (P0) and then amplified by a singlepassage in Vero cells (P1).

Modifications at the Capsid/PrM junction in the DENVax-4b clone did notappear to cause an increase in DENV-4 immunogenicity in NHP studies.Using the sequencing data from the blind serial passages of DENV-4 andDENVax-4b, 3 point mutations that may increase Vero cell adaptation wereidentified. In addition some attenuating mutations were reverted back tothe wild-type sequence to increase immunogenicity in mice. As presentedin FIG. 6, the larger IFU size and lytic phenotypes of DENVax-4e andDENVax-4h illustrates the potential that either of these clones may showan increase in immunogenicity in mice. Experiments to analyze the growthkinetics of each of the new DENVax-4 clones were conducted to determinewhether the inserted modifications increase Vero cell adaptation. Testin A129 mice to analyze immunogenicity are also conducted.

Growth Kinetics

Serial passaging of Dengue vaccine strains in Vero cells is a classicmethod for selecting strains which are better fit to grow in vitro. Inthese exemplary growth experiments, DENVax-2 (FIG. 1) was included as acontrol and displayed the highest initial titer at day 2, followed byDENVax-4b-P10, DENVax-4, and DENVax-4b-P1. At the end of the growthperiod (day 12) DENVax-2 had the highest peak titer, followed byDENVax-4b-P10, DENVax-4b-P1, and DENVax-4.

Amino Acid Changes in Sequences

DENVax4-P10 genomic sequencing demonstrated mutations which correspondedto amino acids E-417 E-K (Glu-Lys) and NS4A-17 M-L. DENVax4b-P10sequencing showed a mutation which corresponded to amino acid C-107 C-Y.The E-417 E-K mutation changes the amino acid residue so that an aminegroup (NH2) is substituted for a carbonyl hydroxyl group. However, the Rgroup is still charged and remains hydrophilic. The NS4A-17 M-L mutationresults in removal of a sulfate from the R group, but maintainsnon-polarity resulting in a hydrophobic amino acid. The C-107 C-Ymutation results in drastic change in the R group. Cysteine has an SHgroup that is capable of forming disulfide bonds, while tyrosine has acarbon benzene ring with a hydroxyl group. This causes the amino acidresidue to become hydrophilic instead of hydrophobic, affecting itsinteraction with the other amino acid R groups.

FIG. 19 is a graphic representation of titers of DENVax-4 constructsduring growth kinetics experiment. The day each sample was taken isplotted on the x-axis and the titer is plotted on the y-axis.

Neutralizing Antibodies in NHP Vaccinated with DENVax

In certain methods, evaluation of the DENV-4 chimeric construct virusestook place with a larger study testing immunization regimens. Theimmunogenicity of DENVax-4b compared to that of DENVax-4a was tested.Groups 1 and 2 were vaccinated on Day 0 with 2 doses and given nobooster vaccination. Equivalent titers of either DENVax-4 (diamonds) orDENVax-4b (squares) were used in all doses. No significant differencesin geometric mean titers (GMT) of neutralizing antibodies were foundbetween any of the serotypes including DENV-4. This suggests that usingDENVax-4b in tetravalent DENVax does affect or increase the neutralizingantibody response against DENV-4. FIG. 20: GMT values comparing Groups 1(DENVax-4, diamonds) and 2 (DENVax-4b, squares). GMT values aremeasurements of neutralizing antibody values from Plaque ReductionNeutralization Technique.

Neutralizing antibody responses in Groups 1 and 3 were compared todetermine whether increasing the dose of DENVax-4 in tetravalent DENVaxincreased immunogenicity against DENV-4. Results demonstrated thatprimates immunized with a higher dose of DENVax-4 showed an increased inGMT of primary neutralizing antibodies detected in the first 60 dayscompared to those immunized with traditional tetravalent DENVax. Therewas no significant difference in GMT between the other DENV serotypes(FIG. 21). This suggests that a higher dose of DENVax-4 improves theneutralizing antibody response against DENV-4.

Neutralizing antibody responses were compared in Groups 1 (diamonds) and4 (squares) to test the effect of the immunization schedule onimmunogenicity (FIG. 22). FIG. 22 illustrates GMT values demonstratingeffect of vaccine schedule on neutralizing antibody response. Thisindicates that 2 doses on Day 0 and no boost has no adverse effectscompared to 1 dose on Day 0 and 1 dose on Day 60. Finally, data fromGroups 4 (diamonds) and 5 (squares) were compared to see if decreasingthe dose of DENVax-2 had an effect on immunogenicity of DENVax-4.Results show no significant difference in GMT against DENV-4, but GMTagainst DENV-2 is significantly reduced in Group 5. This indicates thatthe antibody response elicited by the DENVax-2 dose does not have anadverse effect on the antibody response elicited by DENVax-4 (FIG. 23).FIG. 23 represents GMT values for Groups 4 (diamonds) and 5 (squares)for all DENV serotypes. Results were determined using Plaque ReductionNeutralization Technique.

The results of these experiments support that constructs disclosedherein improve DENV-4 neutralizing antibody responses. IncreasedDENVax-4 in the dose formulation does show a significant increase inneutralizing antibody production. The DENVax-2 dose also does not appearto have an impact on neutralizing antibody responses of DENVax-4. Theremay be little to no difference in antibody production between the prime2 doses vaccination method and prime and boost vaccination method.Sufficient neutralizing antibody titers were produced against DENV-41036, the strain that is currently used in DENVax-4. A successful DENV-4vaccine should be able to adequately neutralize multiple strains of wildtype DENV-4 including newly evolved strains with genome modifications,different genotypes and different phenotypes

During sequencing there were three point mutations identified betweenpassage 1 and passage 10 in both constructs. These mutations werelocated in the capsid region of DENVax-4b and in the prM and envelopegenes of DENVax-4 as previously discussed. Incorporating these mutationsinto the constructs may provide increased growth in Vero cells bydecreasing the attenuation of the virus, which could improveimmunogenicity. As disclosed herein DENVax-4h had a 2-fold increase inneutralizing antibody titers while DENVax-4e had a 1.5-fold increase inneutralizing antibody titers. In other methods, increased DENV-4immunogenicity is tested and compared to other bivalent, trivalent andtetravalent construct compositions.

Materials and Methods Cell Culture

Vero cells are mammalian cells derived from African Green Monkey kidney.The Vero cell line used in the in vitro experiments. Vero cells weregrown at 37° C. in Dulbecco's Modification of Eagle's Medium (DMEM,Mediatech Inc., Manassas Va.) supplemented with 10% Fetal Bovine serum(FBS, Hyclone, Logan Utah), 2% L-glutamine (Hyclone), and 1%Penicillin-Streptomycin (Pen-Strep, Hyclone). To passage the cellsTryple Express solution (Life Technologies, Grand Island N.Y.) was usedto remove the cells from the flask surface.

Viral Infection of Vero Monolayers

Vero cells were seeded on T-75 cm² flasks approximately 48 hours beforeinfection. DMEM supplemented with 10% FBS, 2% L-glutamine, and 1%Penicillin-Streptomycin was used as cell growth medium. Upon cellconfluency, 1 flask was trypsinized using 4 mL of a 0.25% trypsinsolution diluted 1:5 in PBS. Cells were counted to establish an MOI. Twoof the remaining flasks were infected at a predetermined MO1 in 1 mL ofeither DENVax-4-P2 (1^(st) generation DENVax-4) or DENVax-4b-P3 or otherconstruct and diluted in BA-1 diluent (Bovine serum albumin, 1×M199,0.05M Tris-HCL, 1× L-glutamine, 7.5% Sodium bicarbonate, 1× Pen-strep,1× Fungizone). Viruses were adsorbed onto Vero cells for 90 minutes withrocking every 10 minutes to prevent drying of cell monolayers. Afteradsorption 20 mL DMEM supplemented with 5% FBS was added to each flask.Flasks were incubated for 7 days at 37° C.

Viral Harvests and Subsequent Infections: Blind Passage

After a pre-determined period, the CPE was observed on each flask andviral supernatant was harvested and stabilized in 20% FBS for storage at−80° C. Previously seeded confluent T-75 cm² Vero flasks were infectedwith 1 mL of the viral supernatant from the preceding flask. Virus wasadsorbed for 90 minutes with rocking every 10 minutes. After viraladsorption, 20 mL DMEM 5% FBS was added to each flask. New non-infectedcontrol flasks were plated every 7 days. This process was repeated every7 days for 10 subsequent weeks, yielding 10 passages per virus denotedeither DENVax-4-P2-P1 through P10 or DENVax-4b-P3-P1 through P10 orother indicated denotations for the various constructs.

Plaque Titration of Viruses

Samples from DENVax-4-P2 and DENVax-4b-P3 and other chimeric constructswere taken weeks 1, 5 and 10 were plaque titrated to measure titer.Virus samples were serially diluted 1×10⁻¹ to 1×10⁻⁶ in BA-1 diluent.Samples were plaque titrated in triplicate, and 100 uL of each dilutionwas adsorbed to a pre-seeded 6-well plate of Vero cells for 90 minuteswith rocking every 8 minutes. After adsorption, wells were overlayedwith 4 mL of BSS/Agar (NaCl, KCl, NaH₂PO₄—H₂O, glucose, CaCl₂-2H₂O,Mg50₄-7H₂O) solution and incubated for 4 days at 37° C. On day 4 wellswere overlayed with 2 mL BSS/Agar/Neutral Red solution and incubatedovernight at 37° C. Plaques were counted on Days 5, 6 and 7.

Growth Curve Analysis

Growth kinetics of the adapted strains were analyzed by performing agrowth curve on Vero cells. Vero flasks were seeded as previouslydescribed. On day 0 a confluent flask of Vero cells was counted tocalculate the virus PFU needed to infect the flasks at an MOI of 0.001.Flasks were infected with 1 mL of DENVax-4, DENVax-4b-P1, DENVax-4b-P10,or DENVax-2 or other chimeric construct (e.g. DENVax-4e, 4h etc.).Viruses were adsorbed to the monolayers for 90 minutes with rockingevery 8 minutes. After adsorption, 10 mL cDMEM without FBS supplementedwith 1% F-127 was added to each flask and the samples were incubated at37° C. with 5% CO₂. Samples were collected from the supernatant fromeach flask on Day 2 and Days 4-12. Vaccines were harvested by collectingthe entire amount of the supernatant in the flask, and the growth mediawas replaced with fresh cDMEM-F127 on Day 4 and Days 6-12. Flasks werewashed 3 times with PBS during media changes. Samples were stabilized in1×FTA (FTA:15% trehalose, 1% F-127, 0.1% human serum albumin, PBS) andplaque titrated as previously described to determine titer.

After DENVax-4 and DENVax-4b were both blindly passaged 10 times in Verocells, each passage was sequenced to identify mutations between P1 andP10. Sequencing reactions were done on DENVax-4-P2-P1 and P10 andDENVax-4b-P3-P1 and P10 at the CDC. Viral RNA was isolated from virusstocks using a QIAmp viral RNA kit. Reverse transcriptase PCR (RT-PCR)was used to transcribe the RNA into DNA, using primers previouslydesigned by the CDC. The primers are designed from the sequences ofDENV-2 16681 and DENV-4 1036. Approximately 7-9 PCR fragments perconstruct were amplified by RT-PCR. The DNA fragments were thensequenced by Beckman Coulter using an automated sequencing reaction, andaligned for comparison.

Sequencing

After the various constructs are passaged 10 or more times in Verocells, each passage was sequenced to identify mutations. Sequencingreactions were performed on each sample. Viral RNA was isolated fromvirus stocks using a QIAmp viral RNA kit. Reverse transcriptase PCR(RT-PCR) was used to transcribe the RNA into DNA, using primerspreviously designed by the CDC. The primers are designed from thesequences of DENV-2 16681 and DENV-4 1036. Approximately 7-9 PCRfragments per construct were amplified by RT-PCR. The DNA fragments werethen sequenced by Beckman Coulter using an automated sequencingreaction, and aligned for comparison.

Non-Human Primate Study

Cynomolgus macaques are place in different study groups and vaccinatedwith doses of tetravalent DENVax having various DENV-4 constructs. Oneformulation per group can be tested. Formulation 1 can contained a highdose of DENVax with DENVax-4 1^(st) generation, and primates in Group 1are primed with 2 doses on Day 0 and given no boost. Formulation 2contained a high dose of DENVax with a DENV-4 construct included andprimates in Group 2 can be primed with 2 doses on Day 0 and given noboost. A vaccine can be administered sub-cutaneous or ID or by othermethod using a needle or needleless system and syringe. Samples to testfor serology can be taken on days 0, 28, 58, 73, 90, 128 or otherappropriate timing. Neutralizing antibody responses in sera are measuredby plaque reduction neutralization technique.

Plaque Reduction Neutralization Technique

To test for neutralizing antibody production in sera samples, a plaquereduction neutralization assay can be used. Vero 6-well plates areseeded 2 days before inoculation to ensure monolayer confluency. Serasamples were diluted serially two-fold in BA-1 diluent in a 96-wellplate and incubated with dengue virus for approximately 20 hours at 4°C. After incubation Vero wells are inoculated with prepared virus/seradilutions. Samples are adsorbed for 90 minutes with rocking every 8minutes to prevent drying of the monolayers. After adsorption wells areover-layed with 1:1 solution of BSS and agarose, and incubated at 37° C.for 4 days. On Day 4 cells can be over-layed with a 1:1 ratio of BSSsupplemented with neutral red solution and agarose. Plaques visible onwells are counted on for example, Days 5, 6, and 7. A GMT value refersto the average dilution of sera that can neutralize 50% of the virus.This is measured by determining the number of plaques formed in theabsence of sera, dividing that value by 2 (to account for dilution), andnoting which dilution of sera caused plaque formation equal to or lessthan that amount.

The higher immunogenicity of DENVax-4h (envelope mutation) compared toDENVax-4e (capsid mutation) suggest that the DENVax-4 envelope proteincould be optimized. The envelope protein provides epitopes for thegeneration of neutralizing antibodies, and modifying the sequence tooptimize epitope sites for eliciting a strong antibody response mayincrease antibody titer. The envelope mutation in DENVax-4h at position417 is in the conserved portion in the stem region. DENV-4 has adifferent amino acid in this position compared to other flaviviruses.The stem region is in domain III of the E protein where the strongestneutralizing epitope sites exist. Antibodies that bind and neutralizethis site prevent the stem region from fusing with the endosomalmembrane after endocytosis. In fact, position 417 is conserved among theflavivirus family including DENV-1, -2, -3, and WNV. A reversion from Eto K may change the secondary structure to favor a more robust immuneresponse. A further mutation would be to revert back to the conservedcharged Aspartic Acid (D) as seen in the other flaviviruses.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

What is claimed:
 1. A nucleic acid molecule comprising a chimericflavivirus construct comprising nucleic acid sequences encodingnonstructural proteins and one or more structural proteins from a live,attenuated dengue-2 virus and at least encoding one or more structuralproteins from a second dengue virus, the at least second flavivirus isselected from the group dengue-1, dengue-3 or dengue-4 virus wherein thechimeric construct further comprises one or more mutations comprising, amutation in an envelope (E) protein at a position synonymous to aminoacid 417 when the E protein is from the at least second flavivirus; amutation in the capsid protein at a position synonymous to position 107in the attenuated dengue-2, and a mutation in NS4A at a positionsynonymous to position 17 in the attenuated dengue-2.
 2. The nucleicacid molecule according to claim 1, wherein the second flavivirus isdengue-4.
 3. The nucleic acid molecule according to claim 1, wherein themutation in the envelope (E) protein at a position synonymous to aminoacid 417 changes glutamic acid to a positively charged residue.
 4. Thenucleic acid molecule according to claim 1, wherein amino acid position417 is a lysine instead of the glutamic acid.
 5. The nucleic acidmolecule according to claim 1, wherein the mutation in the capsid (C)protein at a position synonymous to amino acid 107 changes cysteine toan aromatic amino acid residue.
 6. The nucleic acid molecule accordingto claim 1, wherein the aromatic amino acid is a tyrosine.
 7. Thenucleic acid molecule according to claim 1, wherein the mutation in theNS4A protein at a position synonymous to amino acid 17 changesmethionine to a basic amino acid.
 8. The nucleic acid molecule accordingto claim 1, wherein the mutation in the NS4A protein at a positionsynonymous to amino acid 17 changes methionine to a leucine.
 9. Thenucleic acid molecule according to claim 1, wherein the attenuateddengue-2 contains a mutation at position 57 in the 5′NCR, a mutation atposition 53 of ns1 and a mutation at position 250 of ns3.
 10. Thenucleic acid molecule according to claim 1, wherein the attenuateddengue-2 contains a mutation at position 53 of ns1 and a mutation atposition 250 of ns1.
 11. The nucleic acid molecule according to claim 1,wherein amino acid positions 102-106 of attenuated dengue-2 aresubstituted with synonymous amino acids of a dengue-4 virus in thesepositions.
 12. A nucleic acid molecule comprising a chimeric flavivirusconstruct comprising a nucleic acid sequence encoding nonstructuralproteins and at least one or more structural proteins from a live,attenuated dengue-2 virus and at least encoding one or more structuralproteins from a second flavivirus, wherein the at least a secondflavivirus is dengue-4 and wherein the attenuated dengue-2 contains amutation at position 53 of NS1 and position 250 of NS3 but does notcontain mutations in NS2A or NS4A or substitutions thereof.
 13. Thenucleic acid molecule according to claim 12, wherein the attenuateddengue-2 contains a mutation at position 57 in the 5′ NCR.
 14. Thenucleic acid molecule according to claim 13, further comprising astructural protein from a third different flavivirus.
 15. The nucleicacid molecule according to claim 14, wherein the third differentflavivirus is selected from the group consisting of West Nile virus,Japanese encephalitis virus, St. Louis encephalitis virus, tickborneencephalitis virus, and yellow fever virus.
 16. A composition comprisingone or more nucleic acid molecules according to claim 1, and apharmaceutically acceptable carrier.
 17. The composition according toclaim 16, wherein the composition further comprises additionaldengue-dengue chimeric constructs.
 18. The composition according toclaim 16, wherein the composition is a divalent, trivalent ortetravalent composition.
 19. The composition according to claim 16,wherein the compositions further comprises other live, attenuatedflaviviruses.
 20. A composition comprising one or more polypeptidesencoded by the one or more nucleic acid molecules according to claim 1,and a pharmaceutically acceptable carrier.
 21. A use of an immunogeniccomposition according to claim 20 in a method for inducing an immuneresponse to dengue virus in a subject.
 22. The use according to claim21, further comprising inducing an immune response to dengue-1,dengue-2, dengue-3 and dengue-4 in the subject.
 23. A kit comprising oneor more composition according to claim 1 and a container.
 24. A live,attenuated virus comprising a nucleic acid chimera according to claim 1.25. A nucleic acid chimera comprising DENVax4e, DENVax4i or DENVax4h.26. The nucleic acid chimera according to claim 25, wherein the chimerais represented by a nucleic acid sequence of SEQ ID NO: 22 or SEQ IDNO:24.
 27. A composition comprising one or more nucleic acid chimerasaccording to claim 25, and a pharmaceutically acceptable carrier.
 28. Ause of an immunogenic composition according to claim 27 in a method forinducing an immune response to dengue virus in a subject.